Microalgae lipid and biomass for biofuel production: A comprehensive review on lipid enhancement strategies and their effects on fatty acid composition

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

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Microalgae lipid and biomass for biofuel production: A comprehensive review on lipid enhancement strategies and their effects on fatty acid composition

T

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Baharak Sajjadia, , Wei-Yin Chena, Abdul. Aziz. Abdul Ramanb, Shaliza Ibrahimc a

Department of Chemical Engineering, Faculty of Engineering, University of Mississippi, MS 38677-1848, USA Department of Chemical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia c Department of Civil Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Microalgae Lipid enhancement Growth rate Lipid productivity Biofuel Fatty acid composition

Renewable energy sources e.g. biofuels, are the focus of this century. Economically and environmental friendly production of such energies are the challenges that limit their usages. Microalgae is one of the most promising renewable feedstocks. However, economical production of microalgae lipid in large scales is conditioned by increasing the lipid content of potential strains without losing their growth rate or by enhancing both simultaneously. Major effort and advances in this area can be made through the environmental stresses. However, such stresses not only affect the lipid content and species growth (biomass productivity) but also lipid composition. This study provides a comprehensive review on lipid enhancement strategies through environmental stresses and the synergistic or antagonistic effects of those parameters on biomass productivity and the lipid composition. This study contains two main parts. In the first part, the cellular structure, taxonomic groups, lipid accumulation and lipid compositions of the most potential species for lipid production are investigated. In the second part, the effects of nitrogen deprivation, phosphorus deprivation, salinity stress, carbon source, metal ions, pH, temperature as the most important and applicable environmental parameters on lipid content, biomass productivity/growth rate and lipid composition are investigated.

1. Introduction The world population in 2050 is estimated to be 1.5 times the current population. Never before the necessity and challenge for sustainable production methods for food and energy was larger than in this century. On the other hand, about 85% of the current national energy needs are met by combustion of fossil fuels i.e oil, natural gas and coal–finite resources that increases concerns about the depletion of fossil fuel reserves and environmental pollution caused by carbon emissions. Therefore, the national energy strategy is changing towards using and getting more benefit from renewable and sustainable sources of energy to achieve energy security in an environmentally friendly manner. Biochar, biodiesel, bioethanol, biohydrogen are a few promising renewable energy sources, most of which are mainly produced from plant residues. As an example, more than 95% of current commercial production of biodiesel (Fatty Acid Alkyl Ester) is produced from edible vegetable oils at the cost of destruction of vital soil resources, deforestation, consumption of large amount of freshwater and

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much of the arable land. The increasing price of vegetable oil and the crisis of food versus fuel are the other problems associated with biofuel generation from plants, trees and oil crops. Accordingly, demand for other possible sources of biofuel has significantly increased. There is a wide range of raw materials for biofuel production. Based on their biomass feedstock, biofuels and biofuel production are classified into four different generations. First-generation biofuels are produced from mostly edible oil seeds, food crops, and animal fats. The main products of this generation include biodiesel, bioethanol, biobutanol. In contrast, second-generation biofuels use lower-value biomass residues such as Nonedible oilseeds, waste cooking oil and lignocellulosic feedstock materials (e.g. forest residues, sugarcane bagasse, cereal straw). In addition to the previous biofuels, syngas is also considered as the product of this generation. The third and fourth generations of biofuels are mainly produced from algae and other algae/microbes respectively. These feedstocks have potential to produce biodiesel, bioethanol, biobutanol, syngas as well as biohydrogen and methane [1]. Microalgaebased oil and biomass have several superiorities over terrestrial

Corresponding author. E-mail address: [email protected] (B. Sajjadi).

https://doi.org/10.1016/j.rser.2018.07.050 Received 26 November 2017; Received in revised form 20 June 2018; Accepted 27 July 2018 1364-0321/ © 2018 Elsevier Ltd. All rights reserved.

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oleaginous crops [2].

Table 1 Comparison of microalgae oil potential with other biofuel feedstocks. Ref: [20,181–194].

• Algae grow quickly. They double every few hours and can be har• • • • •

vested daily. Generally, each unit area of land used for microalgae plant produces 10 times more oil compared to that of a typical terrestrial oleaginous crop. Algae use sunlight, consume carbon dioxide and release oxygen (O2) as they grow. Generally, they are able to photosynthesize up to 2 kg of carbon dioxide per kg of biomass produced. It has been reported that microalgae generate approximately half of the atmospheric oxygen. Different from terrestrial oleaginous crops, microalgae do not require soil fertility and freshwater. They can grow in saline water medium and thus do not compete with other terrestrial crops [2]. They can live and grow in wastewater and purify wastes while producing a biomass and oil suitable for biofuel production at the same time. The plant growth and the energy of terrestrial oleaginous crop depend on seasonal conditions, solar radiation and all other weatherbased changes while algae tolerate extreme weather conditions. The photo-conversion efficiency of terrestrial crops versus incoming solar radiation for terrestrial crops is generally below 1% in temperate climates while the value can increase up to 5% for microalgae biomass.

The oil production efficiency of microalgae versus traditional vegetable oil crops was compared in term of land use in Table 1. As observed, although the oil content of microalgae is strain-dependent and similar to the oil content of terrestrial seeds, there is a significant difference in the overall biomass productivity and resulting oil yield. Besides, microalgae containing 30% oil by weight of dry biomass could yield almost 10 times more oil than palm, which is the most efficient vegetable oil crop. The value significantly increases for those strains that contain higher amount of oil. It is far in excess of what can be generated from palm, soybean and corn, which are currently the global sources of vegetable oil, respectively. Although microalgae-based oil and biomass yields are strain-dependent, significant economical and noneconomical advantages can be reached by using microalgae as biofuel feedstock. However, microalgae oil is estimated to be 3–4 times more expensive than plant oil [3]. Generally, the oil production from microalgae includes three steps: (1) microalgae cultivation (in open ponds or photobioreactors; (2) harvesting/concentration; and (3) oil extraction. Cultivation and harvesting steps are the limiting keys imposing 40% and 20–30% of the cost and energy in microalgal biofuel production, respectively [4,5]. Identifying an optimal balance among these stages is essential to reduce the environmental impacts and costs of the overall process. In this study, different potential organisms are reviewed in terms of biomass and lipid productivity/composition at first. Then, environmental factors e.g. nitrogen deprivation, phosphorus deprivation, NaCl stress, carbon, pH, temperature, and Fe(III) ion, as the most important and applicable strategies to enhance microalgae lipid content are investigated. The main objective of this study is to provide a comprehensive reference on the synergistic or antagonistic effects of environmental stresses on growth rate (biomass production), lipid productivity, lipid fatty acids compositions assisting the researchers to better understand the reactions of microalgae to the environmental factors. (Fig. 1)

Plant

Gal Oil/ Acre

Fat content in seed %

Oil Palm (Elaeis guineensi) Macauba Palm(Acrocomia aculeate) Pequi (Caryocar brasiliense) Buriti Palm (Mauritia flexuosa) Oiticica (Licania rigida) Coconut (Cocos nucifera) Avocado (Persea Americana) Brazil Nut (Bertholletia excelsa) Macadamia Nut (Macadamia terniflora) Jatropha (Jatropha curcas) Babassu Palm (Orbignya martiana) Jojoba (Simmondsia chinensis) Pecan (Carya illinoensis) Bacuri (Platonia insignis) Castor Bean (Ricinus communis) Olive Tree (Olea europaea) Rapeseed (Brassica napus), canola Opium Poppy (Papaver somniferum) Peanut (Ariachis hypogaea) Sunflower (Helianthus annuus) Tung Oil Tree (Aleurites fordii) Rice (Oriza sativa L.) Buffalo Gourd (Cucurbita foetidissima) Safflower (Carthamus tinctorius) Crambe (Crambe abyssinica) Sesame (Sesamum indicum) Camelina (Camelina sativa) Mustard (Brassica alba) Coriander (Coriandrum sativum) Pumpkin Seed (Cucurbita pepo) Euphorbia (Euphorbia lagascae) Hazelnut (Corylus avellana) Linseed (Linum usitatissimum) Coffee (Coffea arabica) Soybean (Glycine max) Hemp (Cannabis sativa) Cotton (Gossypium hirsutum) Calendula (Calendula officinalis) Kenaf (Hibiscus cannabinus L.) Rubber Seed (Hevea brasiliensis)

610 461 383 335 307 276 270 245 230

35.3 28.35 45 19 80 35.3 8–32 66.9 71.6

194 188 186 183 146 145 124 122 119 109 98 96 85 81 80 72 72 60 59 55 55 54 49 49 47 46 37 33 31 28 26

Lupine (Lupinus albus) Palm (Erythea salvadorensis) Oat (Avena sativa) Cashew Nut (Anacardium occidentale) Corn (Zea mays) Microalgae (low oil content) Microalgae (medium oil content) Microalgae (high oil content)

24 23 22 18 18 6275 10,455 14,635

28 60 48–56 71.2 13.5 48 20 30 45–50 53–71 47.3 50–60 10 33 30–40% 27.8–35.3 49.1 40% 35–46 13–20 46.7 48–52 50–70% 34% 10–20 17.7 35 40 17–24% 20% 24(seed) 40(kernel) 7.2–8.2% 20–45 3.1–11.6% 41.7 4 30 50 70

the first link in the oceanic food chain. These microorganisms convert carbon dioxide and water to oxygen and nutrient-rich biomass in the presence of sunlight through photosynthesis process. More than 50,000 microalga species are known to-date, which are categorized with respect to their ultrastructure, biochemical constituents, pigment composition and life cycle. Microalgae are classified into the microplankton (20–1000 µm), nanoplankton (2–100 µm), ultraplankton (0.5–15 µm) and the picoplancton (0.2–2 µm) with respect to their size. Algae are made up of eukaryotic and prokaryatic cells, which are the cells with nuclei and organelles. However, most of the algae (some researchers say almost all) are eukaryotic. DNA in eukaryotic algae is localized within a minutely perforated nuclear membrane and their nuclei are similar to those of higher plants. All eukaryotic algae have intracellular organelle named as chloroplast, which contains photosynthetic lamellae with chlorophyll in which photosynthesis occurs. However, different types of algae have chloroplasts of different shapes with different combinations of chlorophyll molecules (Chlorophyll A, A

2. Basic information about microalgae 2.1. Microalgae cellular structure Microalgae, named also as phytoplankton by biologists are very small single-cell plant-like organisms without leaves or roots. Green microalgae have a diameter between 1 and 50 micrometers, live in water systems such as streams, rivers, lakes and oceans and in fact are 201

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nonpolar lipids. With respect to the metabolic rate, the proportion of these two types of lipids varies along different growth phases of algae. Polar lipids are structural lipids such as glycolipids and phospholipids. These lipids are bound to the organelle membranes such as the thylakoid membranes of the chloroplast [6,8]. Cell membrane that protects a cell and maintains its shape is mainly composed of glycolipids and phospholipids. The bilayer structure of phospholipids in cell membrane consists of a polar hydrophilic head and two hydrophobic fatty acid tails, which may be either off pure saturated tails or in combined with unsaturated tail [9]. Non-polar (neutral) are storage lipids i.e. triglycerides (TAGs), diglycerides (DAGs), monoglycerides (MAGs), free fatty acids (FFAs), hydrocarbons and pigments. Lipids are stored in algae in various ways depending on the algal species, growth phases and environmental growth conditions. Fatty acid compounds differ with algae. Predominantly, polyunsaturated fatty acids (PUFA) comprise the structural lipid fraction, while monounsaturated fatty acids (MUFA) and saturated fatty acids (SAFA) comprise the storage lipid fraction [6,9,10]. In addition to lipids, carbohydrates are the other products of microalgae derived from dark photosynthesis. In dark reactions, carbon dioxide is reduced to carbohydrates by the Calvin cycle [11]. Carbohydrates can be divided into structural components and storage components too. Structural components are found mainly in cell wall (e.g., pectin, cellulose, and sulfated polysaccharides) while storage components (e.g., starch) can accumulate inside or outside chloroplast. However, the metabolism and composition of carbohydrates is not similar in all groups of microalgae [12]. Microalgae carbohydrates can be used for fermentation into ethanol or anaerobic digestion for methane production [12]. Protein synthesis is the most difficult and complex mechanism in all cells. The main steps of the protein synthesis in a cell include amino acid synthesis, peptide chain condensation reaction and modification of the primary protein. Unicellular photosynthetic green algae are most commonly used for protein production as they only require inexpensive salt-based media, carbon dioxide and light for growth.

Fig. 1. A schematic diagram of a) prokaryotic cellular organization, B) eukaryotic cellular organization (Publishing as Benjamin Cummings PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece). Pearson Education, Inc copy Right License (PE Ref. #: 206202).

3. Microalgae taxonomic groups

& B or A & C). The color of microlage (i.e. green algae) relates to the Chlorophyll. Endoplasmic reticulum, mitochondria and Golgi bodies are the other intercellular organs of all eukaryotic algae. In many green, golden, brown and red algae, pyrenoids are present within the plastids. Pyrenoids are the centers for enzymatic condensation of glucose into starch. Vacuole is the other intercellular orange, which is predominantly found in the plant, fungal and algae cells, occupying 30–80% of the cell’s volume. Lipids are mainly stored in vacuoles within the cell [6,7]. In prokaryotes cells, the nuclear materials are not confined by a nuclear membrane and are nearly dispersed throughout the cell. The membrane-bounded plastids, endoplasmic reticulum, mitochondria and Golgi apparatus are absent and the photosynthetic lamellae occur freely in the cytoplasm. Large aqueous vacuoles, are also absent from the structures of prokaryotes cells. Blue-green algae (Cyanophyceae) are the main group of algae which have prokaryotic cells and conduct photosynthesis directly within the cytoplasm, rather than in specialized organelles. The simplistic unicellular/multicellular structure of microalgae enhances their photosynthetic rates, enabling them for effective carbon sequestration and energy production due to rapid lipids accumulation in their biomass.

Among over 50,000 algae species present in the world, either in only aquatic or terrestrial environments, about 4000 species have been identified. Algae are normally categorized based on i) presence or absence of distinct nucleus, ii) composition and relative amounts of different photosynthetic pigments, iii) types of food reserve, iv) cell wall chemistry, v) presence or absence of flagella, vi) number, type, place of origins and orientation of flagella in the motile cells and vii) mode of reproduction and life history. The modern tendency in classification is to use a combination of these characters. Accordingly, Chapman [13] classified the algae as follows:

Prokayotic

Cyanophyta (BlueGreen Algae)

Division 1:

Rhodophyta (RedClass i) Rhodohyceae Algae) Chlorophyta (Green Classes i) Chorophyceae Algae) ii) Charophyceae ii) Prasinophyceae Euglenophyta Class i) Euglenophyceae Chloromonadophyta Class i) Chloromonadophyceae Xanthophycta Class ii) Xanthophyceae (Yellow/Green) (Yellow/Green) Bacillariophyta Class iii) Bacillariophyceae (Diatoms)

Division 2:

Division 3: Division 4:

2.2. Microalgae biochemical composition

Division 5:

Biochemical composition of microalgae compromises four principal groups of molecules: proteins, carbohydrates, nucleic acids and lipids in varying proportions based on algae classes. The most energy-rich compound is lipid (37.6 kJ g−1), followed by proteins (16.7 kJ g−1) and carbohydrates (15.7 kJ g−1). Microalgae contain primarily polar and

Division 6:

202

Class

i) Cyanophyceae (Myxophyceae)

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Division 7:

Chrysophyta (Yellow-Green Algae)

Division 8:

Phaeophyta (Brown Algae) Division 9: Pyrrophyta (Dinoflagellates) Division 10: Cryptophyta

Classes i) Chrysophyceae (Golden or Golden/ Brown) ii) Haptophyceae Classes i) Phaeophyceae

eukaryotic, containing cellulose in the cell wall. Green algae usually have a rigid cell wall made of an outer layer of pectose and an inner layer of cellulose. Most of the members of this class have one or more storage bodies containing starch besides protein located in the chloroplasts. Some of them may store food in the form of oil droplets.

Classes i) Desmophyceae ii) Dinophyceae Classes i) Cryptophyceae

3.5. Prymnesiophyceae Microalgae in the class of Prymnesiophyceae, known also as the Haptophyceae, consist of approximately 500 species. Haptophytes are primarily marine species. Chrysolaminarin, a carbohydrate food reserve, is the major storage product of these species. Similar to the chrysophytes and bacillariphyceae, fucoxanthin gives a yellow-brown to golden-brown color to the cell and lipids of this class.

These species are not equally interesting for biodiesel production. Microalgae belonging to the five classes of Bacillariophyceae, Chlorophyceae, Eustigmatophyte, Chrysophyceae, Haptophyceae (Prymnesiophyceae) and Cyanophyceas are of primary importance for biofuel production (as highlighted). Amongst them, the green algae (Chlorophyceae) taxonomic group includes the most promising species for biodiesel production.

3.6. Cyanophyceae

Bacillariphyceae, also known as diatoms, is the most widely distributed group of microalgae with about 100,000 species is known. They are mostly unicellular, although they can form colonies of various shapes (e.g. zigzag, ribbon, filament, fans and stars). Bacillariphyceae contains chlorophyll-a and chlorophyll-c. It has silicate cell walls. The cells of diatoms contain a high level of fuvoxanthin, a photpsynthetic accessory pigment that gives them golden-brown color. The main storage compounds of diatoms are triglycerides (TAGs) and carbohydrates.

Microalgae belonging to Cyanophyceae are prokaryotic cells with mucilaginous cell walls or sheaths in blue-green color because of the presences of chlorophyll-a and phycocyanin. They possess unicellular, multicellular or colonial cell structures that contain no membrane enclosed nucleus or chloroplasts. More than 2000 species of this class known up-to-date have a different gene structure than the other microalgae. Some of them can absorb atmospheric nitrogen eliminating the need to provide fixed nitrogen for cell growth. This class of microalgae store less amount of lipids compared to other groups, causing them being less attractive for biofuel production. Nevertheless, they could be potential choices for carbon dioxide mitigation and production of novel bio-products.

3.2. Chrysophyceae

4. Lipids in microalgae

The Chrysophyceae involves 1100 species of unicellular algae and have the photosynthetic pigments containing chlorophylls a and c. Their chloroplasts contain a large amount of the pigment fucoxanthin, giving algae their brown color. They live in both marine and fresh water. Similar to the diatoms, the cell walls of Chrysophyceae algae are made of cellulose and pectin materials. Some species of this class also lack cell walls. The golden-brown algae reserve oil droplets and carbohydrates as storage compounds.

Generally, microalgae generate a wide range of lipids including polar lipids, neutral lipids, wax esters, hydrocarbons, sterols and prenyl derivatives such as carotenoids, terpenes, tocopherols, quinines and pyrrole derivatives such as chlorophylls. Polar (structural) lipids mainly contain a high quantity of Poly Unsaturated Fatty Acids (PUFAs). Phospholipids and sterols are the key polar components that make cell membranes, which act as a selective permeable barrier for organelles and cells and participate directly in membrane fusion events. Moreover, some polar lipids may also behave as important intermediates in cell signaling pathways (e.g., sphingolipids, inositol lipids, oxidative products) and contribute in reacting to changes in the environmental parameters. Storage lipids are mainly in the form of triglycerides (TAGs), having a high content of saturated FAs and some unsaturated FAs. Most microalgae accumulate very little TAGs during exponential growth and the main amount of TAGs can accumulate during stationary phase [7,15,16]. Some oil-rich species have the potential to reach high level of long-chain polyunsaturated fatty acids as TAGs. TAGs can be easily catabolized to supply metabolic energy. They are mainly synthesized in the light, accumulated in cytosolic lipid bodies, and then reused for polar lipid synthesis in the absence of light [17]. Analysis on both accumulation of TAGs in Parietochloris incisa as a green microalgae and storage in chloroplastic lipids demonstrates that TAGs do not only play the role as an energy storage product but also have other roles. For example, in the reaction to an abrupt change in the environmental condition to speed up the adaptive membrane reorganization, PUFArich TAGs may endue specific acyl groups to Mono Galactosyl Diacyl Glycerol (MGDG) and other polar lipids [17]. A list of the most common fatty acids (lipids) along with their main specifications, which are normally included in microalgae and used in biodiesel synthesis are provided in Table 1 of our previous manuscript [18].

3.1. Bacillariophyceae

3.3. Eustigmatophyceae Eustigmatophytes are a small group of yellow-green eukaryotic algae with species claimed to be between 20 and 35. In recent classifications, Eustigmatophceae is considered as a separate lingeage in Stramenopile, Heterokontophyta or Ochrophyta. For a long time, they had been considered members of Xanthophyceae. Hibberd and Leedale [14] discovered that these cells are different with Xanthophyceae in having an eyespot outside the chloroplast. They are in pale-green color due to the presence of chlorophyll-a. Eustigmatophytes are unicellular cells and microalgae belonging to this group are mostly small in cell size (2–4 micrometer). They have uni- or bi-flagellate, coccoil to spherical shaped cells containing polysaccharides in cell walls. Eustigmatophytes contain a high quantity of lipid, which is constituted of a large amount of essential polyunsaturated fatty acids. Therefore, some members of the class, especially the marine species of the genus Nannochloropsis, are very promising for biofuel production. 3.4. Chlorophylceae Chlorophylceas forms a major class of green algae, which are made from eukaryotic cells. The grass green color of Chlorophylceas is due to the dominance of chlorophyll-a and chlorophyll-b light harvesting pigments in the cell. To-date, 2650 species of this class, which may be unicellular, colonial or filamentous has been known. Cells are

4.1. Microalgae lipid content The oil content of the most common microalgae frequently used in 203

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Table 2 Taxonomy and properties of the most suitable strains for lipid and biofuel production. Taxonomy

Cellular Structure

Bacillariophyceae

Eukaryotic

Chlorophylceae

Eukaryotic

Marine & Freshwater & Brackish Mostly Freshwater, A Few Marine

Eustigmatophyceae

Eukaryotic

Mostly Freshwater

Chrysophyceae

Eukaryotic

Freshwater & Marine

Prymnesiophyceae

Eukaryotic

Mainly in Fresh Waters

Cyanophyceae Less attractive for biodiesel

Prokaryotic

Freshwater & Marine

Color

Diversity

Chloroplast Content

Storage

Golden-brown

100,000

Lipid & Chrysolaminarin

Green algae

8000

Chlorophyll-a Chlorophyll-c Chlorophyll-a Chlorophyll-b carotenes and xanthophylls Chlorophyll-a

Pale-green or Yellow-green Golden-brown

< 35 1100

Brown algae Due to fucoxanthin blue-green

500 2000

Chlorophyll-a Chlorophyll-c Chlorophyll-c

Starch Protein Lipid & Chrysolaminarin (liquid β − 1,3-glucan) Lipid Carbohydrate Chrysolaminarin (known as α − 1, 3linked carbohydrate)

chlorophyll-a & phycocyanin

5. Environmental effects on microalgae lipids

researches is summarized in Tables 2 and 3. As observed, oil content of 30% is quite common in most of the species while some have much higher oil content (56% in Nannochloris sp., 53% in Chlorella sp. and 65% in Neochloris oleoabundans). However, higher oil strains grow slower than low oil strains [19]. Besides, there are algae (like Schizochytrium sp.) with more than 80% of their total mass is lipid. It is the oil (fatty acid) that can be extracted and converted into biofuels. However, it has been reported that microalgae containing 80% oil grow 30 times slower than those containing 30% oil [20]. As per Table 3, Chlorophyceae contains the most number of promising species for algaoil production. The microalgae belonging to the Labyrinthulomycetes have been less studies; however, some of the members of this class (i.e. Schizochytrium) can store a high amount of oil. On the other hand, the species of Cyanophyceae and Rhodophyceae are more suitable for protein and carbohydrate production.

It is well known that the biological activities of microalgae highly depend on the culture conditions. Unfavorable conditions could change the cellular composition of microalgae (in phytoplankton cells as an example), their growth rate, lipid accumulation etc. For example, the culture conditions imposed onto the cells may increase the oil content of the Chlorella vulgaris by almost two or three times. Nutrient availability, e.g. nitrogen, phosphorous, and/or silicate (in diatoms) in the culture medium is the key to the growth and primary production of microalgae. The supply of carbon source, salinity, light intensity, and temperature are among the other effective factors. Overwhelmingly, compared to other nutrients, nitrogen and phosphorous manipulations have been found to be the most effective factor to increase lipid accumulation several folds in microalgae, respectively. Theoretically, the most common elements in the medium of alga culture should follow the stoichiometric formula of C106H181O45N16P to reach the optimal growth of algae. Low ratio of nitrogen to phosphorus of about 5:1 suggests Nlimitation and high ratio of about 30:1 suggests P-limitation. The sensitivity of microalgae to nutrient manipulation is usually considered through three levels of limitation including starvation, limitation and depletion. During starvation, microalgae are grown in nutrient replete environment first and then the cells are separated and transferred into another media with the absence of a particular nutrient. The lack of nutrient causes a sharp biological shock resulting in generation and saving of high-energy compounds. Nutrient limited growth involves growing organisms in continuous cultures with a media in which all nutrients are available in excess except one particular nutrient that limits the maximum quantity of biomass that can be produced and causes physiological reaction to the limiting nutrient. The basic idea of this method is named as ‘‘law of the minimum’’ in microbiology. It assumes that there is always one particular nutrient that specifies the utmost amount of biomass that can be produced. Nutrient deficient growth is commonly performed in batch cultures. This method involves growing cells in an environment replete with required nutrients. The growth rate of the organisms rise along with cell density until the nutrient depletion is achieved. Then, the growth rate and photosynthesis potentially reduce while high-energy storage compounds increase due to some alterations in the metabolic processes in response to repletion of required nutrients.

4.2. Micro algae lipid composition The amount and ratio of saturated and unsaturated fatty acid is a key that determines the suitability of microalgae as a biofuel feedstock. The relevant details of the most widely used microalgae-oil are summarized in Table 4. Generally, unsaturated fatty acids, especially palmitolleic (16:1), oleic (18:1), linoleic (18:2), linolenic acid (18:3) and the saturated fatty acids of palmitic (16:0) are the main compositions of the oil generated by microalgae with only a small proportion made from saturated fatty acids of stearic (18:0). Some microalgae can also synthesize a large quantity of polyunsaturated fatty acids such as C22:6 (42%) in Aurantiochytrium sp., C22:5 + C22:6 (39.4%) in Schizochytrium limacinum, C20:5 (25%) in Porphyridium cruentum. The fuel properties of biodiesel produced from microalgae significantly depend on the composition of miacroalgal fatty acids. For example, the biodiesel generated from the microalgae fat with lower saturated fatty acid content presents better cold temperature properties because long-chain saturated fatty esters dramatically increase the pour and cloud point of biodiesel. However, biodiesel containing a high amount of unsaturated compounds gets oxidized faster than conventional diesel, leading to insoluble sediments to interfere with engine performance. Therefore, the ability of microalgae in generating a high quantity of lipid and high quality of the fatty acid compositions should be considered while seeking efficient microalgae strains for biofuel production. Besides, most microalgae species are able to thrive under extreme conditions, in which their growth rate, lipid content and fatty acid composition are significantly affected. Understanding the effects of such situations on microlalgae behavior gives us a controlling parameter to achieve more favorable results.

5.1. Nitrogen factor 5.1.1. Effect of nitrogen on lipid content of microalgae Protein is the major macromolecular pool of intracellular nitrogen and mainly affected by nitrogen rather than any other nutrients e.g. phosphorus [21]. Nitrogen is an essential constituent of cell structure 204

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Table 3 Oil content of marine microalgae expressed on a dry matter. Class

Edible Vegetable Oil

Oil Content (%)

Protein (%)

Carbohydrates (%)

Bacillariophyceae

Phaeodactylum tricornutum Thalassiosira weissflogii Skeletonema costatuma Chaetoceros muelleri

18–57 5–20 13–51 13–24

30 43 25 31–43

8.4 12 4.6 7–28

Dunaliella primolecta Dunaliella tertiolecta Dunaliella. salina Dunaliella Bioculata Nannochloris sp. Nannochloropsis oculata Scenedesmus obliquus Scenedesmus quadricauda Scenedesmus dimorphos Scenedesmus sp. Ankistrodesmus sp. Ankistrodesmus fusiformis Chlamydomonas reinhardtii Chlamydomonas sp. Parietochloris incisa Tetraselmis tetrathele b Neochloris oleoabundans Scenedesmus falcatus Scenedesmus protuberans

23 11–16 6–25 8 20–56 22–29 30–50 1.9 16–40 17–24 11.48–31

< 64% 20–29 57 49 16.69 35 10–45 40–47 8–18 29–37 16.24–18.66

11–23 12.2–14 32 4 – 7.8 20–40 12 21–52 32.7–41 4.48–5.97

21 22.7 62 25–30 35–65 6.41–9.6 17.53–29.30

48 58.8 – – 10–27 3.37–7.83 25.4–45.05

17 18.5 – – 17–27 2.73–6.83 20.95–29.21

Chlorella Chlorella Chlorella Chlorella Chlorella Chlorella Chlorella

28–53 41–58 2 40–60 23–63 22–24 14–57

25–45 51–58 57 10–28 36 40.5 47.89

24–30 12–17 26 11–15 41 26.8 8.06

Nostoc commune Synechocystis sp.d Spirulina Platensis Spirulina Maxima Spirulina sp

22 11 4–11 6–7 7.72

20.3–43 63 46–63 60–71 57.47

34–56.4 15 8–14 13–16 23.9

Pavlova lutherie Pavlova salinaf

35 12–30

29 26

9 7.4

Emiliana huxleyi

43.8

39.2

17.2

Heterosigma akashiwo

43

50

7

Chroomonas salina

12–14.5

29–35.5

9–11

Porphyridium cruentumc

9–14

28–39

40–57

Mesotaenium.sp.

19–35

53

27

Schizochytrium limacinum Schizochytrium sp. Aurantiochytrium sp.

43 50–77

39 15

5 12

Chlorophyceae

Eustigmatophyceae sp. vulgaris pyrenoidosa protothecoides emersonii sorokiana minutissima

Cyanophyceae

Haptophyceae

Prymnesiophyceae Raphidophyceae Cryptophyceae Rhodophyceae Conjugatophyceae Labyrinthulomycetes

*Not belongs to this class but belongs to this phylum. a: Phylum: Bacillariophyceae, Class: Mediophyceae; b: Phylum: Cholorophyta, Class: Chlorodendrophyceae; c: Phylum:Rhodophyta, Class: Porphyridiophyceae; d: Phylum: Cyanobacteria; Class: Porphyridiophyceae; e: Phylum: Haptophyta; Class: Pavlovophyceae; f: Phylum: Haptophyta, Class: Pavlovophyceae, g: Phylum: Haptophyta, Class: Prymnesiophyceae. Ref:[195–215].

Different strain species develops different strategies to cope with variations in cellular N quotas, which can either make differences in rates of protein turnover or rearrangement in intracellular pools [22]. With respect to the temporal changes of N, some algal cells regulate N supply and some others regulate energy supply for N assimilation. The production of organic storage compounds is an example of the latter strategy. After transferring from N-sufficient culture to N-depleted culture, cell proteins decreased within the first few days (3–4 days depend on the species) due to continued cell division followed by dilution of cellular pools by new biomass production. Therefore, cell

and functional processes of microalgae since it is a key component of proteins, amino acids, nucleic acids, enzymes and photosynthetic pigments. It also affects the lipid content and growth of algae. Moreover, cellular nitrogen and lipid content are inversely related and so analysis of organic nitrogen is a representing key for nitrogen starvation and the beginning of lipid induction. Organic nitrogen per biomass of < 3% is usually considered as the nitrogen starvation. Under sufficient nitrogen concentration, the molar rate of photosynthetic carbon fixation is 7–10 folds the rate of nitrogen assimilation, which is an appropriate ratio for synthesis of essential nitrogen-containing cellular components. 205

Bacillariophyceae Phaeodactylum ricornutum Thalassiosira weissflogii Skeletonema costatum Chaetoceros muelleri Chlorophyceae Dunaliella primolecta Dunaliella tertiolecta Dunaliella. salina Nannochloris sp. Nannochloropsis oculata Scenedesmus obliquus Scenedesmus quadricauda Scenedesmus dimorphos Scenedesmus sp. Franceia sp. Ankistrodesmus sp. Ankistrodesmus fusiformis Chlamydomonas reinhardtii Chlamydomonas sp. Parietochloris incisa Tetraselmis viridis Eustigmatophyceae Chlorella sp. Chlorella vulgaris Chlorella pyrenoidosa Chlorella protothecoides Chlorella salina Cyanophyceae Nostoc commune Synechocystis sp. Synechocystis sp. PNN 6803 Spirulina Platensis Spirulina Maxima Spirulina sp Haptophyceae Pavlova lutheri Pavlova salina Emiliana huxleyi Raphidophyceae

Edible Vegetable Oil

–

–

–

–

– 6.9

0.8

0.04

8.3

–

2.4 – – –

–

– – –

3.6 0.7 1.18 –

–

– – –

1.0 0.5

– – –

–

–

–

– 2.43

0.5

0.13

15.2

–

0.58 – – –

–

– – –

– – – –

–

– – –

– – –

– – –

C 12:0

–

C 10:0

Table 4 Fatty acid composition of pure edible vegetable oils.

206 10.95 13.1 30.3

0.9 1.5 3.28

0.3 27.9 2

3

2.8 1.91 0.5 0.8

– 0.7

3.07

0.46

0.4 0.6

0.5

1.57

0.413

0.5 0.77 0.97 4.95 9.1

15.59

12.3

13.4

7.2

C 14:0

– – –

– – 3.8

– – –

–

1.6 1.58 – 0.8

– – –

–

0.03

– 1.5 –

1.12 – –

– – –

–

1.56 0.9 0.45 –

– – 2.6

– – –

1.96 – –

– – –

–

0.7 1.8 0.9 –

– – –

–

– 2.2 – –

– 0.6 – – –

2.3

0.4

– 2.0

–

–

9.4

–

– –

–

–

–

–

C 15:1

0.36

0.97

3.1 0.63 2.51

1.14

2.7

–

0.7

C 15:0

1.92

2.65

5.94

– –

–

–

–

–

C 14:1

4.13

24.8

17.4 15.1 14

41.4 37.7 39.7

34.4 22.7 56

22.7

18.3 15.3 18.6 16.2

51.9 13.5 16.1

17.8

17.9 12.9 16.2 25.3

15.8

27.3 30.4 5.5

3.44 3.44 6.68

17.7 36.9 4.05

6.9

3.48 2.63 2.67 5.07

1.2 2 5.9

5.77

3.9 7.3 3.06 0.34

5.2

3.62

2.7 2.59 3.53 10.6 26.1

23.9 26.1 14.4 18 35.1

28

37.8

19.9

31.8

35.8

C 16:1

24.6

7.25

32.4

18.6

C 16:0

1

2.84 – –

0.4

–

3.51 6.3 3.63 5.8

– 3.1 2.15

–

– 1.5 – –

2.1

2.58

0.62

0.9 1.72 1.3 1.02 –

3.06

3.75

–

2.93

C 16:2

– 0.5 –

0.05 – –

– – –

–

8.76 4.9 4.49 –

– 1 1.25

–

– 2 – –

2.1

–

3.63

2.5 3 3.8 2.9 –

3.4

13.6

–

8.3

C 16:3

– – –

– – –

– – –

–

0.3 – – –

– – 19.1

–

– 17 – –

15.6

–

11.2

–

12.3 17.4 20.4

–

7.8

7.4

1.4

C 16:4

– – –

– – –

– – –

–

2 1 6.45

2.32 3.42 3.66

1.5 3.5 2.4

8.69

2.46 3.37 2.54 2.89

21.1 6.2 0.85

6.85

1.9 0.5 7.18 1.46

0.6

4.84

1.31

1.2 4.05 2.77 1.84 2.53

2.06

1.15

1.5

1.53

C 18:0

(continued on next page)

– 6.47 – –

– – –

–

– –

1.7

10.7

0.4

– –

–

–

–

–

C 17:1

B. Sajjadi et al.

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

–

–

–

–

–

– –

–

–

0.1

–

–

– –

C 18:1

Edible Vegetable Oil

207

11.6 2.3

11.3

6.93

37.5 11.1

25.6

23.9

8.9

10.2 6.7 17.7 65.2

Nannochloris sp. Nannochloropsis oculata Scenedesmus obliquus Scenedesmus quadricauda Scenedesmus dimorphos Scenedesmus sp. Franceia sp. Ankistrodesmus sp. Ankistrodesmus fusiformis

– – – 1.02

– – 0.65 –

– – 2.55

– –

30.9 33.3 28.7 3.84

20.7 8.5 8.48 2.24 3.7 – –

0.12

–

–

26

12.8

3.5

–

–

6.7

1.61 –

–

–

0.21 –

–

–

7.85

4.28

3.2 0.1

–

C 20:1

– –

–

2.3

–

–

–

–

–

–

C 15:1

–

C 20:0

– –

5.1

0.5

0.3

–

1.4

2

0.4

C 15:0

2.32

14.4

13.5 0.98

1

38.4

11.5

11.6

Dunaliella. salina

2.35 1.3

6.6 22.9

11.4 8.6

39.9 41.4

1.0

1.81

2.85 0.8

0.5

1.4

1.55

1.28

C 18:4

– –

–

–

–

–

–

–

–

–

C 14:1

0.9

0.3

1.33

9.8

2.13

C 18:3

5.5 3.03

3.94

0.5

0.5

5

12.4

Bacillariophyceae Phaeodactylum ricornutum Thalassiosira weissflogii Skeletonema costatum Chaetoceros muelleri Chlorophyceae Dunaliella primolecta Dunaliella tertiolecta

5.64

0.5

–

–

C 18:2

–

–

–

6.4

–

–

Heterosigma akashiwo Dinophyceae Gymnodinium kowalevskii Cryptophyceae Chroomonas salina Rhodophyceae Porphyridium cruentum Chromalveolata Amphidinium sp. Trebouxiophyceae Picochlorum sp. Conjugatophyceae Mesotaenium.sp. Labyrinthulomycetes Schizochytrium limacinum Schizochytrium sp. Aurantiochytrium sp.

C 14:0

C 12:0

C 10:0

Edible Vegetable Oil

Table 4 (continued)

– – – –

0.8

4.42

2.56 0.1

0.26

–

–

0.1

–

0.2

C 20:2

14.8 40

46.6

13.4

16.8

29.6

34.4

13.5

26.7

43.2

C 16:0

–

–

– – – –

–

0.17

– 0.9

–

–

0.03 4.57

– –

– 0.4

5.53

20.8

14.1

18.4

0.05

–

– –

–

2.2

5.1

–

0.2

0.2

4

C 16:2

C 20:5

0

– 1.91

–

–

–

–

C 20:4

– 0.66

–

6.1

1.2

1

2.28

2

2

17

C 16:1

– – – –

–

–

2.72

0.01 –

9.44

– –

–

–

–

–

C 22:1

– –

–

2.4

3.5

–

0.2

–

–

C 16:3

– – – 0.01

–

–

–

0.4 –

5.12

1 –

0.68

3.5

–

1.33

C 22:6

– –

–

16.4

–

–

–

–

0.4

C 16:4

0.48d 3.18 f 36.4c

9.97a

3.7c

Other

– –

[202] [227] [202] [231]

[227]

[216] [208,217,222,223] [207,209,217,224] [216,225] [217,226,227] [203,228,229] [204,230]

[217,221]

[217,220]

[216,219]

[16,216–218]

Ref

– 1

3.6

0.6

3.4

3.5

0.8

3

8.5

0.5

C 18:0

(continued on next page)

3.5

–

–

–

–

–

–

–

C 17:1

B. Sajjadi et al.

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

13.2

20.9

13.8

15.3 38.5

11.3

12.6

16.7

17.6 10.3

27.6

Chlorella vulgaris

Chlorella pyrenoidosa Chlorella protothecoides Chlorella salina

208

17.5 20.1 17.6

15.5 13.8 16.2

7.86 9.01 6.17

– –

– –

– –

– –

– 0.4

–

–

–

1

7.1

–

–

3.1

31.4

11.8

7.6

–

2.1

–

– –

–

– –

–

–

–

–

–

14.9

35.8

15.5

2.3

1.9

–

3.7

0.3

0.37 – –

– – –

–

– –

1.05

0.4

– – 0.95

–

C 20:2

0.1

5.7

1.1

11

0.55

–

0.2

19.1

0.25

1.33

–

–

–

5.2

15.6

–

–

–

10.8

–

6.5

6.3

– – –

– – –

– – –

–

7.2

3.1

2.7

9.1 4.2 6.85

– 2.52 3.33

– – –

1.5

– 2.35

1.85

0.26

– – 1.05

3.8

C 20:1

30.3

5.5

0.6 – 0.9

8.8 3.8 32

0.5 2.2 5.5

27.2 9.45 14.6

15.9 0.2 16.

6.9 8 6.6 – – –

–

–

29.7

– – –

– –

– –

14.4 26.5

– – –

3.12

12.9

–

– – –

– – 12.7 3.43

–

–

0.1

C 20:0

C 18:4

Total C22:5 + C22:6, a: C24:1, b: C22:5, *C22:5 +C22:6, c:C8:0, d: C22:0, f: C22:2.

Haptophyceae Pavlova lutheri Pavlova salina Emiliana huxleyi Raphidophyceae Heterosigma akashiwo Dinophyceae Gymnodinium kowalevskii Cryptophyceae Chroomonas salina Rhodophyceae Porphyridium cruentum Chromalveolata Amphidinium sp. Trebouxiophyceae Picochlorum sp. Conjugatophyceae Mesotaenium.sp. Labyrinthulomycetes Schizochytrium limacinum Schizochytrium sp. Aurantiochytrium sp.

Cyanophyceae Nostoc commune Synechocystis sp. Synechocystis sp. PNN 6803 Spirulina Platensis Spirulina Maxima Spirulina sp

9.03 5.07 15.7

10.4 16.3 –

15.9 20.4 7.35

16.0

6.58

32.4

Chlamydomonas reinhardtii Chlamydomonas sp. Parietochloris incisa Tetraselmis viridis Eustigmatophyceae Chlorella sp.

C 18:3

C 18:2

C 18:1

Edible Vegetable Oil

Table 4 (continued)

– 0.6

–

–

–

–

27.6

2.8

–

3.5

0.3 3.7

– – –

– – –

–

– –

1.06

1.69

– 30 0.25

–

C 20:4

– 0.6

–

0.8

–

14.5

25

12.9

0.1

11.8

15.1 19.1

– – –

– – –

–

0.24 –

0.47

1.3

– 2.65 6.15

–

C 20:5

– –

–

–

–

–

–

–

–

– –

– – –

– – –

–

– –

– 42.6

–

–

–

–

–

–

9.5

–

9.7 – –

– 9.97 –

– – –

–

– –

–

–

– –

– – –

–

C 22:6

– – –

–

C 22:1

10b

39.4 *

2.1c

2.2c

Other

[242,243] [244,245]

[240,241]

[227]

[227]

[227,239]

[215]

[217]

[217]

[216]

[216] [217] [216]

[236,237] [236–238] [195,215,237,239]

[216] [216] [214]

[210,211,217,235] [202–204,208,235,236] [208,236] [202,205,208] [202,208,212,213]

[233] [216,234] [217]

[202,232]

Ref

B. Sajjadi et al.

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

accumulation in microalgae. For example, some authors have got benefit from temperature manipulation to reach proper balance of growth and lipid content in N-deficient conditions [28]. In an analysis on Nannochloropsis, Fakhry et al. [28] found that the combined effect of nitrogen and temperature stressors created an additional response by not only increasing cellular lipids and triglycerides but also enhancing lipid productivity in a shorter duration. However, this solution cannot be generalized to all algae because the lipid production and productivity are specific for each strain and subject to many factors. For example, although the lipid content in the Nannochloropsis [28], eustigmatophyte N. salina [29] and chrysophytan Ochromonas danica [30] increases with temperature, there is no significant response to the temperature factor in the lipid content of Chlorella sorokiniana [31]. Nitrogen also has a great interactive effect with other cultural parameters that are specific for each strain. In an investigation on the combined effect of nitrogen and phosphorus deprivation with iron, Singh et al. [32] found that nitrogen under the lowest concentration of iron (3 mg L-1) had a negative effect on both the lipid content and productivity of Ankistrodesmus falcatus whereas its effect changed positively as the iron concentration increased to its moderate value (6 mg L-1). In another analysis on B. protuberans and Botryococcus braunii in batch cultures, Singh et al [33] reported that Botryococcus spp. under anaerobiosis and nitrogen supplemented medium produced less amount of lipid than under anaerobiosis and nitrogen deficient conditions. Light and pH are the other factors that may have synergistic or antagonistic effects on lipid content of a species under nitrogen deprivation condition. Breuer et al. [34] found that the highest TAG content of Scenedesmus obliquus under nitrogen deficient conditions was independent of light intensity, but it varied from 18% to 40% of dry weight, depending on temperature and pH. It reached the maximum yield of fatty acids at the lowest light intensity, 27.5 °C and pH 7. Some researchers have also focused on the effect of cultivation duration under nutrient limitation. Shorter normal growth time may result in low oil accumulation, while long normal growth time may lead to cell disruption, followed by low oil production. Cao et al. [35] reported that the lipid content of Chlorella minutissima rose up to 29.19% of the dry weight of algae after 6-day normal condition and 3 days with limited nitrogen. Similar phenomenon was also observed during the cultivation of Chlamydomonas reinhardtii, which reached the maximum oil synthesis between 2 and 3 days following nitrogen depletion followed by a plateau around day 5 [36]. However, the proper length of normal nutrition culture causes variations in lipid content and should be analyzed as priority. And the last but not the least point, algae are capable of utilizing nitrite, nitrate, ammonia and urea as the sources of nitrogen sources. As also observed in Table 6, most studies use nitrate in nitrogen depletion process, while urea has been more used in large-scale algal cultivation because of its relative low cost and universal availability compared to the others. However, the control of urea concentration during the cultivation is a basic challenge. Urea can release urease or be hydrolyzed to ammonia in alkaline conditions. As a result a portion of the nitrogen source is wasted, and the ammonia toxicity inhibits the growth at high levels [37]. Many reports have indicated that Spirulina platensis can use ammonium, nitrate and urea as nitrogen sources for growing [37–39]. While the use of nitrate as nitrogen source for Neochloris oleoabundans results in a higher growth rate and lipid content than that of using urea but the cell grow poorly in mediums with ammonium as the nitrogen source [40]. A nitrogen source test on Chlorella sorokiniana has also demonstrated that this strain can grow well with urea and nitrate, but not ammonium [41]. In the opposite, Ellipsoidion sp. with ammonium grow faster and accumulate higher lipid than that with urea and nitrate [42,43]. It has been reported that urea is a favorable nitrogen source for green algae growth [44]. Arthrospira (Spirulina) platensis [37] and Chlorella [43] demonstrate an excellent ability to uptake urea as nitrogen source for their growth. Danesi et al [39] reported that urea addition in an intermediate form had a favorable effect on the growth rate of Spirulina platensis at 27 °C while

divisions slow down while producing energy storage products is mobilized. In the initial phases of nitrogen deficiency, carbon fixation may exceed carbon demand for nitrogen assimilation and extra carbon may be converted into lipids and carbohydrates as the main storage compounds before photosynthetic capacity is dramatically exhausted. In other words, the ability of microalgae to use fixed carbon is switched from protein and polar lipids synthesis toward either carbohydrate or storage oil generation. Generally, nitrogen limitation imposes three changes to the species: decreasing the cellular content of thylakoid membrane, activation of acyl hydrolase and stimulation of the phospholipid hydrolysis. These changes may increase the intracellular content of fatty acid acyl-CoA. Meanwhile, nitrogen limitation could activate diacylglycerol acyltransferase, which converts acyl-CoA to triglyceride (TAG). Therefore nitrogen limitation could increase both lipid and TAG content in microalgal cells [23]. If N is resupplied, storage compounds supplies the energy and C for N assimilation until photosynthetic capacity is restored [24]. Nitrogen limitation results in a relative increase in carbohydrate and/or lipid storage of microalgae and a decrease in protein content, photosynthetic efficiency and growth rate [22]. However, these responses in microalgae are species-specific. For example, Chlorella sp. showed a great reduction in the protein content while no difference was observed in Prorocentrum donghaiense cultured under various nutrient limitations [21]. Generally, the lipid content of green algae multiplied up to 2- to 3-fold due to nitrogen deprivation for 4–9 days, whereas both increase and decrease have been reported in diatoms, depending on the species [25]. However, in an analysis on the effect of nitrogen starvation on lipid accumulation in chlorella Rathinasabapathi et al. [26] reported that lipid accumulation in algal cells began just hours after they were starved of nitrogen – not days. The oil content in some of the most important microalgae under nitrogen limitation is summarized in Table 5. As observed in some of the previous studies, low nitrogen medium induced lipid accumulation up to 30–70% within 7–20 days in most microalgae, which increased their calorific value [27]. Monallantus salina, reached the highest concentration of lipids (72%) well into 9 days of deficient nitrogen growth. Botryococcus braunii, Chlorella vulgaris and Nannochloropsis sp. have demonstrated the potential to reach 61.4%, 57.9% and 55% lipid production during their life frame, respectively. Some other microalgae, like Scenedesmus sp. and Chlorella sorokiniana have not reached a high content of lipid even during 7 and 14 days. However, as the average value, most of them reached to > 40% of lipid production under nitrogen deficient condition. As already mentioned, the increased oil content of algae due to nitrogen manipulation comes at the cost of diminished growth rate (see Table 5, in case of Chlorella minutissima for example). In other words, the overall rate of lipid generation is diminished during nutrient deficiency phase and so culturing either few cells with high lipid content or many cells with low lipid content will not produce an economically viable biofuel feedstock. Hence, there should be a favorable tradeoff between lipid accumulation and growth. With respect to this challenge, Adams et al [24], investigated this relationship in six species of green algae under high and low levels of N deficiency and reported the following trend for the productivity of those species: Chlorella vulgaris > Chlorella oleofaciens > Neochloris oleoabundans > Scenedesmus dimorphus > S. naegleii > Chlorella sorokiniana. They discussed that the most promising microalgae species as biofuel feedstock are the ones, which grow and accumulate lipid concurrently and respond to little stress that does not significantly damage the growth rate. In other words, Chlorella vulgaris and Chlorella oleofaciens significantly respond to low stress while Chlorella sorokiniana, Neochloris oleoabundans, Scenedesmus dimorphus and S. naegleii are more sensitive to high stress. Accordingly, the lipid productivity, which reflects the independent effects of N supply on both growth and lipid content should be the benchmarked. Some researchers have also analyzed the effects of nitrogen manipulation combined with other cultural conditions on lipid 209

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

Table 5 The effect of nitrogen limitation on microalgae lipid, lipid productivity and growth rate. Nitrogen Source

N-stress

Nitrate NaNO3 Nitrate

N-Deficiency N-Deficiency N-Deficiency 0.025–0.105 g/l N-Deficiency N-Deficiency 1.5–0.375 gL N-Deficiency to

KNO3 NaNO3 NaNO3 aerobic NaNO3+ KNO3 NaNO3 anaerobic KNO3 anaerobic KNO3 aerobic NH4Cl KNO3 NaNO3+ KNO3

NaNO3 urea ammonium NaNO3+ KNO3 NaNO3+ KNO3

NaNO3 anaerobic NO3–N

NH4Cl KNO3 NaNO3 NaNO3 NaNO3 NaNO3 aerobic

N-Starvation 0.45 g/l N-Deficiency 10%X-1.25%X N-Starvation 1.76 mM N-Deficiency 0.15–0 g/l N-Starvation 10 mM N-Deficiency 0.1% N-Deficiency No-stress, Low stress:11 mM N High stress:4 mM N N-Deficiency 3 mM(limited) 20 mM (rich) N-Starvation 0.45 g/l N-Deficiency No-stress, Low stress:1mM N High stress:4mM N N-Deficiency 10%X-1.25%X N-Deficiency+ P-Deficiency 0.01 g/l N-Deficiency 0.1% N-Deficiency 0.247 g/l N-Deficiency 1.5–0.375 gL N-Deficiency N-Deficiency 0.075 g/l

-

N-Deficiency

KNO3 Nitrate NO3 NaNO3 anaerobic NaNO3+ KNO3

N-Deficiency,0.4 g/l N-Starvation N-Deficiency N-Deficiency 10%X-1.25%X N-Deficiency No-stress, Low stress:11 mM N High stress:4 mM N N-Deficiency 10%X-1.25%X N-Starvation

NaNO3 anaerobic -

1.76 mM

Microalgae Species

GR-ND g L-1d

GR-NS g Ld

days

Ref

1

-

1.21µ -

−0.1 µ -

-

150

-

-

15 7 8

[22] [7] [66] [246]

55% -

10.01

16.41

0.13µ

0.1µ

10 14

[247] [46]

52

230–25 °C

370–25 °C

0.61B

0.48B

12

[28]

-

5

[248]

-

7

[249]

10

[250]

10 8 10

[58]

LC-NS (%)

LC-ND (%)

LP-ND mg/ Ld

LP-NS mg/L d

Dunalliela tertiolecta Dunalliela. tertiolecta Dunalliela. tertiolecta Nannochloropsis sp.

12.9 36 1x -

20.3 42 2.3x ~50–53%

-

Nannochloropsis sp. Nannochloropsis oculata Nannochloropsis salina

30

Nannochloropsis sp.

28.7

56%

90

-

2.5

Nannochloropsis sp.

35

59

80

35

-

Nannochloropsis oculata Scenedesmus obliqus

22

31

-

-

Scenedesmus obliquus

53

-

33 58.3b 40%, > 250 h

Scenedesmus obliquus Scenedesmus obliquus Neochloris oleoabundans

0 21.2 13

34.6 45.6 29-58

Neochloris oleabundans

9 29

37 16 17 50%

Neochloris oleabundans

12

a

B

0.07µ a

B

0.03µ

0.22

-

585.9 2160b -

-

-

-

-

[251]

91–131

0.033B 0.018B 1.9B

-

-

0.186B 0.403B 2.4B

12

[24]

40 90

150 65 70 -

2.7B -

1.85B -

7

[40]

5

[248]

12

[24]

7

[249]

12

[53]

B

Scenedesmus dimorphus S. naegleii

9

20-34

-

86–111

5.3

10

21-39

-

83–83

4.8B

Scenedesmus sp.

20

22

-

-

-

0.1

B

4.0

B

[34]

2.0B B

0.18

B

Scenedesmus sp

25

30

150

50

0.67

Chlorella vulgaris Chlorella vulgaris Chlorella vulgaris

0 22.6 15.5

52.8 57.9 24.6

-

-

[251]

31.5

0.038B 0.017B 1.28B

-

29

0.205B 0.287B 1.87B

10

[252]

Chlorella vulgaris

-

-

8.16

20.30

0.14µ

0.13µ

14

[46]

Chlorella vulgaris Chlorella vulgaris Chlorella emersonii Chlorella protothecoides Chlorella sorokiniana Chlorella minutissima Chlorella luteoviridis Chlorella capsulate Chlorella pyrenoidosa Chlorella pyrenoidosa Chlorella minutissima Chlorella sp. Chlorella sp.

29.53 18 29 11

40 40 63 23

12.77 41 28 2.5

8 37 79 23

0.99µ 0.86µ 0.33µ

0.77µ 0.46µ 0.27µ

17 14 14 14

[95] [253]

20 31 11% 12.5

2.7 32

4.8 16

0.58µ 0.43µ

0.19µ 0.43µ

14 14 -

[253]

-

-

0.315 -

0.127 -

23

22 57 28.8 11.4 29.2 26% 29.19 30-50% 21

-

-

-

-

12+12 6+3 7-12 7

[254] [35] [255] [249]

Chlorella sorokiniana

15

21-47

-

68–85c

4.1B

1.8B

12

[24]

Chlorella vulgaris Chlorella oleofaciens Tetraselmis suecica

10 12 25

40-48 35-48 35

-

146–94 127–86 -

4.3B 4.3B -

2.1B 2.0B -

7

[249]

Tetraselmis subcordiformis Pavlova viridis

13.5

25

-

-

0.04µ

0.012µ

10

[250]

25

26

0.065µ

0.015µ

(continued on next page)

210

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

Table 5 (continued) Nitrogen Source

N-stress

Nitrate

N-Deficiency

Microalgae Species

KNO3 aerobic

N-Starvation 0.4–0 g/l

KNO3

N-Deficiency

NO3

N-deficiency to N-Free for 10 days N-deficiency to N-Free for 10days N-Deficiency 0.037 g/l N-Deficiency 0.1%

NO3 NO3 aerobic KNO3 KNO3 KNO3 KNO3 NaNO3 N-free NaNO3 NH4Cl urea

N-Deficiency, 0.75 g/l -

LP-ND mg/ Ld

LP-NS mg/L d

-

-

GR-ND g L-1d

GR-NS g Ld

days

Ref

1

0.84µ

0.1µ

15

[22]

LC-NS (%)

LC-ND (%)

Thalassiosira pseudonana Thalassiosira pseudonana Botryococcus braunii Botryococcus protuberans Botryococcus (TRG) Botryococcus (KB) Botryococcus (SK) Botryococcus (PSU) Green Alga Diatom Monallantus salina

11.5

25.9

21

31

38.6 35.2

61.4 52.2

-

-

-

-

10 10

[33]

25.8 17.8 15.8 5.7 17.1 24.5 -

35.9 d 30.2 28.4 14.7 30-50 ~ 72%

46.9 39.7 21.3 3.5 -

-

0.182µ 0.223µ 0.135µ 0.061µ -

−0.07µ −0.11µ −0.05µ −0.1µ -

7C 14S

[70]

4–9

[25]

-

-

-

-

9

[25]

Isochrysis zhangjiangensis Anacystis nidulans Microcystis aeruginosa Oscillatoria rubescens Spirulina platensis Ankistrodesmus falcatus Ellipsoidion sp.

-

2.32fold

-

-

-

-

4

[256]

14.8 23.4 12.8 21.8 23.33 -

14.3 17.7 0 0 59.6 7.99 27.6 33.3 21.5

-

-

[251]

74.07 -

0.008B 0.011B 0.05B 0.065B 1.74B -

-

58.99 -

0.453B 0.136B 0.289B 0.235B 3.54B 0.17µ 0.29µ 0.31µ 0.28µ

14

[32] [42]

[7]

P: Productivity [mg L−1 day−1], C: Control Condition, S: Stress Condition, dcw: of dry cell weight. a: 1.5% glucose-supplemented condition, b: under optimized condition: N:0.04 g/l, P:0.03 g/l, c: The values of productivity correspond to the low and high stress, d: A combination of nitrogen deficiency, moderately high light intensity (82.5 μE m-2 s-1) and high level of iron (0.74 mM), ~: both decrease and increase observed NS: Nitrogen Sufficient, ND: Nitrogen deficient, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate

in some microalgae, the reduction of other saturated compounds is of importance; (C23:0 and C24:0 in Chlorella sp.). Nitrogen stress has been found to cause a reduction in the saturated fatty acids of Ankistrodesmus. falcatus [32], which could improve the Cetane number and oxidative stability of the biodiesel produced. On the other hand, no significant change in the amount of saturated fatty acids of C. vulgaris but a slight decrease in those compounds of N. Oculata have been observed [46]. The same completion is observed in terms of monounsaturated values between C16:1 and C18:1 compounds. The balance between these compounds causes the increment of MUFA in Chlorella. Vulgaris and Scenedesmus obliquus, but a slight reduction in Chlorella sp. Linolenic acid is the main poly-unsaturated compound in most microalgae lipids. The presence of linolenic acid methyl ester (C18:3) in high amount, above 12% based on European standards (EN14214) leads to lower stability of biodiesel due to oxidation. However, the percentage composition of linolenic acid is slightly reduced in some microalgae e.g. A. falcatus, grown with nutrient stress, making it suitable for biodiesel production. While no significant changes were observed in C18:3 of C. vulgaris and N. Oculata [46]. Very few studies can be found in case of nitrogen sources rather nitrite and nitrate. Based on Fidalgo’s work [47], the urea usage as the

continuous mode presented more benefit to both growth and lipid content at higher temperature (30 °C). Comparing the effects of nitrate, nitrite and urea on the lipid content of Isochrysis galbana, higher lipid content can be yielded by urea if it is added in logarithmic manner or during early stationary phase while nitrite and nitrate are more favorable if they are added at the late stationary phase. Besides, although nitrate is widely used in nitrogen depletion process, microalgae grown in wastewater first metabolize ammonia before nitrate [45]. Accordingly, research combining ammonia depletion with the reclamation of water would have been beneficial not only to oil and biofuel synthesis but wastewater treatment industries. 5.1.2. Effect of nitrogen on lipid composition Aside from the quantitative effect of nitrogen on lipid content, its qualitative effect is of importance for biofuel production (Table 7). Although the major fatty acid compositions in microalgae are similar in both control and nutrient-stress medium, there is a considerable difference in their percentage composition. Nitrogen stress has been found to decrease the saturated fatty acid of C16:0 while increase C18:0. Accordingly, the final effect of nitrogen depends on the tradeoff between this increase and decrease. However,

Table 6 The effect of urea limitation on microalgae lipid, lipid productivity and growth rate. Nitrogen Sources

Worst Conc.

Best Conc.

No* or Neg

Microalgae Species

L-LC %

H-LC %

L-LP g/Ld

H-LP g/Ld

L-GR g L-1

H-GR g L-1

day

Ref

Urea, 0.1–0.3g l-1

0.15

0.25

< 0.25 <

16.64

18.25

38.3

49.29

–

–

12

[44]

0.025 0.56 5.6 –

0.1 1.1 2.5 –

< 0.1 < < 1.1 < – –

Desmodesmus abundans Chlorella sp. Spirulina platensis Spirulina platensis Isochrysis galbana

0.66 – 20.7 –

0.52 – 18.6 42.05

0.051 – – –

0.124 – – –

0.036 870 dcw 0.47 –

0.058 1270 mg l− 1 0.61 –

6

[43] [38] [39] [47]

Urea, Urea, Urea, Urea,

0.025–0.2g l-1 0.56–1.7 mM 0–5.6 g 4 mg atom Nl−1

L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate. Worst and best concentrations versus Lipid content and Lipid Productivity. 211

18 14

212

[66] [32] [251] [251] [251] [251] [251] [251] [250] [250] [250] ↑ ↓ ↓↓ ↓ – ~ ~↓ ↓ ↑ ↓ ↓ ↓ ↓ ↑↑ ↑↑ ↓↓ ~ ~↓ ~↓ ↓ ↑ ↑ – – – – – – –

↓ ↑ ↑ ↑↑ ↑↑ ~ ~↑ ~ ~↓ ↑ ↑ – ↑ –

– ↑ – – – – – – ~ – – – – – – – – – – ↑ ~↑ ↑ ↑ ↓ ↓↓ ↓↓ – ~ ~ ↑ ↑ – ↑ ↓ ↓ ↓ ↓ – ↑↓ ~↓ ~↓ ↓ – ↑ ↓ ↓ ↑↑ ↑↑ ↑↓ ~↑ ~↓ ~↓ ↓ ~↑ – ~ ↑ ↑ ~↑ ~↑ ~↑ – – ~ – – – – – – – – – – – – – – – – – – – – – – – – ↑↓ ↑↓ – – – – – – – – ↑↓ ↓↓ – – – – ↑ ~ – ~ – ~ ↓ – – – – ~ ↑ ~↓ – ~ ↓ ↓↓ ↓↓ ~↓ ~ ↑↓ ~ ↑ ↑ ↓ ↑ ↑ ↑↑ ↑↑ ~ ~↑ ~↑ ↓ ↑ ↑ – – – – – – – – – – – – – – – – – – – – ↑ – Dunaliella tertiolecta Ankistrodesmus falcatus Scenedesmus obliquusb Scenedesmus obliquus Anacystis nidulans Microcystis aeruginosa Oscillatoria rubescens Spirulina platensis Tetraselmis subcordiformis Nannochloropsis oculata Pavlova viridisc

Chlorella vulgaris Chlorella vulgaris Nannochloropsis.oculata

a: C23:0: ↓, C24:0: ↓↓, b: C18:4: ↓, C: C22:5: ~↓.

–

– – – – – – – – ~ ~↑ ~↑

– – – – – – – – – – –

↓↓ ↓↓ ↑ ↑↑ ↑↑ ↓ ~↓ ~↑ ↓ –

– – – – – – ↓↓ ↓↓ ~ ↓↓ ↓↓ ↑↑ ↑↑ ↑↑ ↓ ~↑ ~↑ ↓ – – – – – – – – – ↑↓ ↑↓ – ↓↓ ↓↓ – ↓ ↓ – ↓ ~↑ ↓ – – – – – – – – –

~ – ↓ – ~ –

Starvation 1.5–0.37 Limitation NH4Cl KNO3 0.3–0.07 Limitation NaNO3 deficiency deficiency NH4Cl deficiency KNO3 deficiency KNO3 deficiency KNO3 deficiency KNO3 deficiency KNO3 deficiency deficiency deficiency deficiency Chlorella sp.a Chlorella. vulgaris

~ –

– – –

[65] [46] ↑ ~↓ ↓ ↑ ~ ~ ~ – ↑ – ↑↑ – ↑ ~↓ ↑ ↓ ↓ ↑ ↑↑ ~ – – – – – – – – – –

C 17:0 C 16:4 C 16:3 C 16:2 C 16:1 C 16:0 C 15:1 C 15:0 C 14:1 C 14:0 g/l Edible Vegetable Oil

Table 7 The effect of nitrogen stress (compared to normal growth condition) on fatty acid composition of different microalgae.

C 17:1

C 18:0

C 18:1

C 18:2

C 18:3

C 20:0

C 20:1

5.2.1. Effect of phosphorous on lipid content of microalgae Phosphorus is the other essential nutrient for the growth of microalgae as it plays a key role in cellular metabolic processes, which are related to signal transduction, energy transfer and photosynthesis. Phosphatases convert the organic phosphates to orthophosphates at the cell surface and microalgae take up the phosphorous as inorganic orthophosphate (PO43–). This process requires energy and occurs especially when inorganic phosphate is in short supply. In high supply, microalgae assimilate excess phosphorus in their cells in the form of polyphosphate granules so they can prolong the growth process in the absence of phosphorus. Accordingly, the growth of microalgae does not immediately respond to the environmental changes of phosphorus concentration, in contrast to the instant responses of microalgae to light and temperature. It has been reported that the phosphorus concentration in cells varied with supply concentration, from a minimum of 10 mg dry mass per mg P at supply concentration of 5 mg P.l–1 to a maximum of 1170 mg dry mass per mg P at a supply concentration of 0.1 mg P.l–1 [48] (Table 8). Under phosphorus starvation, a drastic reduction in membrane phospholipids happens and these compounds are replaced by nonphosphorus glycolipids and sulfolipids, offering an effective phosphorusconserving mechanism as it has been reported for Chlorella sp. [21]. Li et al. [49] discussed that galactolipid, which is a type of glycolipids, was accumulated to rectify the loss of phospholipids under phosphorusdeprived conditions. These changes represent an effective phosphateconserving mechanism. Besides, cell division rates are reduced under Plimited condition, though photosynthetic rates slightly decreased. This may result in accumulation of carbon, which might be stored in the form of triacylglycerols that are rich in saturated fatty acids and monounsaturated fatty acids [50]. Spijkerman et al. [51] reported that the total FA content increased over twofold, increasing cellular C content while total FA content decreased with increasing cellular P content over a factor of ten. However, it has also been reported that a decrease in the cellular P content results in an enhanced FA accumulation, independent of cellular C status. In addition to lipids, carbohydrates are considered as a long-term store under nutrient limitation. In other words, depending on the species, microorganism could accumulate either lipid or carbohydrate or both. In phosphorus deficient environments, more lipids, mainly TAG, can be stored in Dunaliella parva [52], Chlorella sp. [21] Scenedesmus sp. [53], Monodus subterraneus [54], Chaetoceros sp. (Badllariophyceae), Pavlova lutheri (Prymnesiophyceae) [55] while carbohydrates have higher storage proportion in Arthrospira (Spirulina) platens [56] Nannochloris atomus (Chlorophyceae), Tetraselmis sp. (Prasinophyceae) [55]. In contrast, protein as the major macromolecular pool of intracellular nitrogen, has been found to be mainly affected by nitrogen rather than phosphorus. In an effort to identify the optimum condition between phosphorus and nitrogen as the two most determining nutrient compounds, Xin et al. [53] reported that N/P should be controlled in a proper range of 5:1–8:1 to increase the lipid content of Scenedesmus sp. Although they could reach higher amount of lipid but lipid productivity and accumulation rate were not at their highest. Similar value of N/P (8:1) was reported by Kapdan and Aslan [57] for Chlorella vulgaris too. It has also been reported that Scenedesmus obliqus can reach to the lipid content of 58% at the nitrate, phosphate and thiosulphate

~ –

C 20:2

5.2. Phosphorous effects

~ ~

SFA

MUFA

PUFA

Ref

nitrogen source resulted in generation of lipid with higher content of polyunsaturated fatty acids (C18:4, C20:5, C22:6), while nitrite and nitrate increased the proportion of saturated (C14:0) and monounsaturated compounds (C16:1). The addition mode of urea also demonstrated a great effect on fatty acid composition of the intercellular microalgae. The least amount of saturated and monounsaturated but highest amount of polyunsaturated compounds obtained by adding the urea at the early stationary phase.

[251] [251] [46]

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

concentrations of 0.04, 0.03 and 1 g/l, respectively [58].

5.3. Metal effects

5.2.2. Effect of phosphorous on lipid composition Phosphorus limitation causes dramatic changes in the biosynthesis process and hence the composition of the lipid. As per Table 9, the fatty acid composition of algae is consistent with their taxonomy. Unsaturated fatty acid, which makes the highest proportion of lipids in green algae Chlorella kessleri, significantly increases under phosphorus starving conditions [59]. The same result has also been reported for Chlamydomonas acidophila [51] and Chlorella sp. [60], P. tricornutum, Chlorella sp., I golbaria [61]. It was because although the fatty acid composition of lipids is species-dependent, the membrane lipid classes in the green alga are mainly constructed by unsaturated fats such as 16:3, 16:4, 18:1, 18:2, 18:3 and 18:4 and an enhanced level of unsaturated fatty acids cause greater membrane ‘‘fluidity’’ [51]. However, it has been reported that the concentration of 16:4(3,7,10,13) and 18:3(9,12,15) is more sensitive to carbon source (their concentration in low CO2 cells is higher) and less to P-limitation [61]. However, opposite results have been observed for Ankistrodesmus falcatus under the combined effects of nitrogen, phosphorus and iron. Singh et al. [32] discussed that decrease in percentage of polyunsaturated fatty acid and simultaneous increase in the composition of saturated fatty acid under nutrient stress condition could be possible because of the oxidative damage of unsaturated fatty acids. The same results has also been reported in case of the green alga Scenedesmus quadricauda, which has demonstrated a lower percentage of polyunsaturated fatty acids at P-limited than at P-replete conditions, mainly due to the lower content of 18:3(9,12,15) [62].

5.3.1. Effect of metals on lipid content of microalgae The trace metals have a significant effect in the growth rate, lipids and carbohydrates content in numerous microalgae. However, based on the analysis in different cases, it can be concluded that the effectiveness of these compounds depends on their concentration in the media, their interactive synergy or antagonistic effects with other environmental factors and species type (Table 10). Iron is one of the most effective trace metals because ferric ion is involved in fundamental enzymatic reactions of photosynthesis. Iron is crucial to regulate the gene expression and metabolism in algae. Presence of Fe3+ in the culture media prolongs the period of the exponential growth phase and increases the final cell density although it cannot promote lipid accumulation in exponential growth phase. It has been reported that the exponential growth phase in the presence of Fe3+ is at least 4 days longer than those in the absence of Fe3+[63,64] while the change in cultures with NaNO3 supplement was opposite to this [64]. Accordingly, iron deprivation expects to reduce the biomass growth. For example, Praveenkumar [65] analyzed the effect of iron deprivation on Chlorella sp., and stated that under this situation, there was a slight decrease in biomass with no significant increase in lipid content. The same results have been reported in the case of Dunaliella tertiolecta but with a significant reduction in the cell growth [66]. Iron limitation could also increase the content of glucose, as reported in Agmenellum quadruplicatum from 5% to 45% [67]. In some microalgae, increasing the bio-available iron concentration in the culture medium could result in a simultaneous increase of both growth rate and lipid content [68]. However, excessively high or low Fe3+ in the culture medium has an inhibitory effect on the growth and lipid production (Table 10).

Table 8 The effect of phosphorus limitation on microalgae lipid, lipid productivity and growth rate. Phosphate Source

P-Stress

Microalgae Species

Taxonomy

C-LC (%)

S-LC (%)

C-LP mg/Ld

S-LP mg/Ld

C-GR g L-1d

S-GR g L-1d

days

Ref

Na2HPO4 Na2HPO4 PO4–P -

P-Deficiency P-Deficiency P-Deficiency P-concentration 4–0.5 mM C-1 P-concentration 40–0 mg L-1 P-Starvation N-Starvation

Dunaliella tertiolecta Scenedesmus obliqus Scenedesmus sp Chlamydomonas acidophila

G G G G

1x 7 22 8

0.9x 24 53 30

90 -

75 -

0.1B 0.35B -

~↓ 0.8B 0.14B -

7 10 12 1.6

[66] [58] [53] [51]

Ankistrodesmus falcatus

G

31.31

59.6

54.79

74.07

0.175B

0.124B

14

[32]

Choricystis minor

G

B

PO43-

P-concentration Normal:1.7 mM Limited:0.017 mM

G G G

NaH2PO4

P-Deficiency Low stress: 50%μmax High stress: 5%μmax

K2HPO4

P-Starvation 175–0 μM P-Deficiency P-concentration 240–32 μM P-Starvation

Coccomyxa mucigena Coccomyxa peltigera variolosae Trebouxia aggregata Trebouxia erici Nannochloris atomus Tetraselmis sp. Phaeodactylum tricornutum Chaetoceros sp. Isochrysis galbana Pavlova lutheri Gymnodinum sp. Monodus subterraneus

G G G GBb GBb Br BrH Di YGx

Chlorella sp. Chlorella sp. Chlorella kessleri

K2HPO4 BG11 medium

K2HPO4 K2HPO4 Na2HPO4, NaH2PO4 KH2PO4

P-concentration 22–444 mg L-1

Botryococcus braunii

25.2% 27% 26%

34.2% 45.3% 59.5% ↓↓ ↓↓ ↓↓

-

TAG↓ TAG↑ TAG↓

B

1.45 1.89B 1.85B -

1.3 1.86B 1.73B -

2 5 10 14

[257]

0.83–0.078 0.38–0.046 0.53–0.047 0.66–0.006 0.54–0.051 0.53–0.061 0.30–0.15 1x

4–5

[61]

4+4

[54]

[258] and [55]

-

TAG↓↓ -

6.5a

↓ 11-9 15-8 12-20 11-21 22-30 21-23 13-12.5 39.3a

-

-

1.66 0.82 1.06 1.29 1.11 1.19 0.55 3.4x

YG YG

31.2 14

31.9 23.6

40.27 6

39.35 15.67

2.59B 1.9B

2.45B 2.2B

16+4b 22

[65] [21]

YG

7.4

9.5

-

-

-

-

6

[59]

30

[68]

C

P

-

22% at 444 mg L-1

54% at 222 mg L-1

-

-

0.8

B

1.9

B

a: TAG, b: Normal Condition: 16 days, deprivation condition: 4 days, P-concentration: Analyzing different concentration of P, GBb: Golden-Brown Bacillariophyceae, YGx: yellow Green Xanthophyceae, BrH: Brown, Phylum Hyptophyta, PC: Phylum: Chlorophyta, C: Control Condition, S: Starvation Condition, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate B: Biomass Production and rest: Specific Growth rate, 213

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

– – – –

– – – – ↓ – – – – – –

↑↑ ~ ~ – ↓↓ ~ ↓↓ ↑ ↑↑ ~ ~

↓↓ ~↓ ↑ – ↑ ↑↑ ↑ ↓ ↑↑ ↑↑ ↑↑

↓ ↑ ↓ – ↓ ~↑ ↓ ↑ ↓ ~↑ ~↓

↓ – – – ↑ – ~ ↓ ~ – –

– – – – – ↓ – – – –

↑ – – – – –

↑ – – – ↑ g D – C e f

↑↑ ~↑ ~ ↑ ↓↓ ↑ ↓ ~ ↑↑ ↑ ↑

↓↓ ↓ ~ ↑ ↑ ↑↑ ↑↑ ↓ ↑ ↑↑ ↑↑

↓ ↑ ↓ ↑ ~ ↑ ↓ ↓ ↓↓ ↑ ↑

[32] [258] [258] [51] [21] [61] [65] [59] [54] [61] [61]

Generally, most studies have reported that Fe3+ concentrations up to almost 2 × 10-3 g/L have a positive effect on the microalgal growth while Fe3+ concentrations beyond that concentration (up to almost 4 ×10-2 g/L) affect microalgal growth negatively. However, the lipid content increases with a greater amount of Fe3+ up to 1 × 10-3 while further increase in Fe3+ (1 ×10-2 -1 ×10-1) concentration does not improve the lipid accumulation or reduce it as reported for in Scenedesmus sp. [69], Botryococcus [70], Scenedesmus dimorphus [68], C. vulgaris [64]. In case of Chlorella vulgaris, it has been reported that the total lipid content in cultures supplemented with 1.2·10-5 mol L-1 FeCl3 is up to almost 3–7 folds than that in other media supplemented with lower iron concentration [64]. This result has also been confirmed by Baky et al. [71] in the case of Scenedesmus obliquus. The authors reported that the maximum biomass production was achieved at 10 mg/L Fe3+ and beyond that a decreasing rate was observed. However, the highest lipid content and lipid productivity were observed at 20 mg/L Fe3+ [71]. The highest lipid content in Chlorella sorokinian was observed with 10−4 mol l−1 of iron which was 2.8-fold than that without iron [63]. In addition to iron, appropriate concentration of calcium and magnesium [72], cadmium, copper and zinc can also be favorable for the algal biomass and lipid production. Magnesium has demonstrated an important role in the growth of microalgae so that Mg starvation hinders cell division [73]. It is discovered that the increase of Mg2+ could promote Acetyl-CoA carboxylase (ACCase) in vivo activity. ACCase can catalyze and increase the production of microalgal cells [69]. The lipid content and productivity of microalgae are much enhanced when Mg2+ concentration is in the range of 2 × 10-3-8 × 10-3 g/L. The role of calcium is more critical in the signal transduction of environmental stimuli. Neutral lipid synthesis can be regulated based on cytosolic Ca2+ level through Ca2+ signals in microalgae [74]. Moreover, Ca2+ concentration in the level of 5 × 10-4–5 × 10-3 g/L is also associated with the rise in lipid content in hetrotrophic cultivation of Scenedesmus sp. [69] and the photoautotrophic cultivation of C. vulgaris [72]. Compared to iron, magnesium and calcium demonstrate a stronger effect on lipid content and productivity of Scenedesmus in different concentrations [69]. It has been found that the lipid productivity of Scenedesmus under Mg2+ concentration of 7.3 × 10-3 g/L is about 390% higher than that of the control. Heavy metals such as cadmium, copper and zinc are also known to alter the lipid metabolism of algal resulting in enhancement of lipid content in some microalgae e.g. Euglena gracilis [83,84] and algal lichen [85]. Higher concentration of fatty acids has also been observed in C. vulgaris (0.2 mg/g), C. protothecoides (0.16 mg/g) and Chlorella pyrenoidosa (0.11 mg/g) at copper levels of 4 mg/L [75]. Copper stress (31.4 mg/L~approximately 0.49 mM) can yield lipid accumulation of 5.78 g/L in Chlorella protothecoides [76]. Silica deficiency is the most common stress in diatoms promoting storage lipid accumulation in this taxonomy. Silica effect is more rapid and severe than N or P deficiencies in these organisms and can provide a controllable means to induce lipid synthesis in a two-stage production process. As an example silicon limitation increases the lipid content in diatoms of Chaetocerosmuelleri, Cyclotellacryptica, and Navicula saprophila, up to almost 89%, 110%, and 104%, respectively [77]. Although most studies have focused on the effect of individual metals in microalgae behavior, few studies have investigated the combined impacts of metals with other parameters on improving biomass and lipid productivities of microalgae. It has been found that Ankistrodesmus falcatus can reach its maximum lipid content and lipid productivity under moderate nitrogen (750 mg/L) and high iron supplementation (9 mg L−1), while hosphorus show no significant influence on lipid productivity [32]. Generally, a greater improvement in the lipid content is demonstrated when the Fe3+ is sufficiently supplied in the medium under nitrogen-deficient condition [70]. Combined influence of iron/nitrogen/NaCl and iron/nitrogen/potassium-phosphate positively affects the lipid accumulation in Chlorella minutissima [35]

a: Nitrogen and phosphorus starvation, N: not assessable due to the interactive role of copper and lead. b: ↑: C16:1 ω7, ↓: C16:1 ω9. c: ↑: C20:3 ω6, ↓: C20:4 ω6, ↓↓: C20:5 ω3. d: ↑: C24:0. e: ↑: C20:5. g: ↑: C20:4, ↑: C20:5. f: ~↑: C18:4, ↑: C22:6.

– – – – ↑↑ – – – – – – – – – – – – – – – – – – – – – – – – ~ – – – – – – – – – – ↑↑ – – – ~ ↓ ↓ – ~ ↑ ↑↑ ↓ ↑↓b ↑ ~ ↑↑ N ↑ – ~ ↑ ~ ↓ ~ ↑ ↑ – – – – ~ – ↑ – – – – – – – – ↓ – ~ – – – – – – – – ↑↑ ↑ ~ ~ ~ ↑ ↑ Ankistrodesmus falcatusa Coccomyxa mucigena Coccomyxa peltigera variolosae Chlamydomonas acidophila Chlorella sp. Chlorella sp. Chlorella sp. Chlorella kessleri Monodus subterraneus Phaeodactylum tricornutum Isochrysis galbana

– – – – ↑ ~ ↑ – – ~ ~

C 16:3 C 16:2 C 16:1 C 16:0 C 15:1 C 15:0 C 14:1 C 14:0 Edible Vegetable Oil

Table 9 The effect of phosphorus stress (by reduction) on Fatty acid composition of different microalgae.

C 16:4

C 17:0

C 17:1

C 18:0

C 18:1

C 18:2

C 18:3

C 20:0

C 20:1

C 20:2

SFA

MUFA

PUFA

Ref

B. Sajjadi et al.

214

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Table 10 The effect of metal availability on microalgae lipid, lipid productivity and growth rate. Iron Source

Worst Conc.

Best Conc.

FeCl3 0–20 mg L−1 FeCl3·6H2O 0–0.4 mM FeCl3·6H2O 0–1.2e-5 M FeCl3·6H2O,0–1e-4M

0

20

< 20

0

0.05–0.10 mM

< 0.05–0.10 <

0

1.2e−5

–

0

1e−4

< 1e−4

Iron deprivation FeCl3-6H2O,0.86 ppm

0 0

normal 0.93

– –

MnCl2-4H2O

0

927 ppm

–

Na2MoO4-2H2O

0

1.45 ppm

–

ZnCl2

0

0.028 ppm

–

3+

-4

−3

−3

1.2e < 7.3e−3 < 9.8e−4 < 1e−3 <

Algal Species

L-LC(%)

H-LC(%)

L-LP mg/ld

H-LP mg/ld

L-GR mg/ ld

H-GR mg/ ld

day

Ref

Scenedesmus obliquus Chlorella minutissima Chlorella vulgaris

5.75

28.13

20.1

95.35

[71]

16.73

[35]

56.6

0.140 gld –

7

7.8

0.120 gld –

1.25B mg/ l –

18

11.25

0.891B mg/l –

[64]

12.5

32.5

–

–

18e6 Cell/ ml 0.46B

25

Chlorella sorokiniana Chlorella sp., Dunaliella tertiolecta Dunaliella tertiolecta Dunaliella tertiolecta Dunaliella tertiolecta Scenedesmus sp. Scenedesmus sp. Scenedesmus sp. Scenedesmus sp. C. vulgaris

13e6 Cell/ ml 0.4B

23

[63]

B

B

16 7

[65] [66]

Fe , 0–1.2e g/l Mg3+,0–7.3e-4 g/l Ca3+,0–9.8e-4g/l EDTA,0–1 g/l Mg,0–0.1 g L−1 Ca, 0–0.02 g L−1 NaCl (0–0.05 g L−1) Mg 0–0.1 g L−1 Ca 0–0.02 g L−1 NaCl (0–0.05 g L−1) FeSO4,9–45mgL−1

0 0 0 1 0 0.02 0.05 0 0.02 0.05 45

1.2e 7.3e−3 9.8e−4 1e−3 0.1 0 0 0.1 0 0 27

< < < < –

CuCl2 0.05–0.22 mM ZnCl2 0.22–1.76 mM Chromium 0–96.4 μM Chromium 0–96.4 μM Combined effect Iron, Ni, Pi (NH4)5Fe(C6H4O7)2 3–9 mgL−1 Fe3+, 0–0.74 mM Ni Rich-deficient Fe3+, Acetate 0.1–20 μM

control

0.22

–

Botryococcus braunii Euglena gracilis

control

0.88

–

Euglena gracilis

96.4 μM

48.2

96.4 μM

48.2

– – – < 27 <

S. obliquus

Euglena gracilis UTEX Euglena gracilis MAT

2.58 –

31.2 1x

31.4 0.52x

39.96 –

40.27 –

2.54 –

1x

0.42x

–

–

–

–

7

[66]

1x

0.42x

–

–

–

–

7

[66]

1x

0.42x

–

–

–

7

[66]

9 35 11 26 15.1 20.8 11.9 14.9 20.0 11.3 11

42 42 48 45 27.1 40.3 39.1 26.4 37.0 38.7 35

0.03 0.04 0.065 0.04 –

0.245 0.245 0.280 0.280 –

–

–

–

0.8 μg/ 105cell 0.8 μg/ 105cell 2.2 μg/ mg 2.7 μg/ mg

1.7 μg/ 105cell 2.1 μg/ 105cell 4.2 μg/ mg 4.5 μg/ mg

– B

B

3.4 3.5B 3.5B 3.5B 1.4B 0.9B 1.02B 1.4B 0.8B 0.98B 0.22B

6 6 6 6 18 18 6 18 18 6 –

[69] [69] [69] [69] [72]

–

1.3 0.7B 3.5B 0.7B 0.6B 1.4B 1.2B 0.5B 1.2B 1.1B 0.25B

–

–

–

–

3

[259]

–

–

–

–

3

[260]

–

–

–

–

6

[81]

–

–

–

–

6

[81]

[72]

[68]

9

Ankistrodesmus falcatus

47.96

59.6

25.35

74.07

0.053B gld

0.124B gld

14

[32]

Fe: 0 Ni-rich

Fe: 0.74 Ni-deficient

–

29 36 15 30 –

– – – – –

– – – – –

[261]

0.1

20

–

Chlamydomonas reinhardtii

–

–

–

–

9

[261]

Fe3+, 2.4e−5,4.8e−5 M CO2:0.036% Fe3+, 2.4e−5,4.8e−5 M CO2:1% Fe3+, 2.4e−5,4.8e−5 M CO2:2%

4.8e−5

2.4e−5

–

Chlorella vulgaris

17

20

–

–

– – – – 1.68 µ (day) 0.5e7B 0.74 µ (day) 7e6B 0.22B

9

Fe3+,CO2 0.1–20 μM

– – – – 0.96 µ (day) 4e6B 0.56 µ (day) 1e6B 0.25B

[70]

20

11 13 5 11.5 –

20

0.1

Botryococcus (SK) Botryococcus (TRG) Botryococcus (PSU) Botryococcus (KB) Chlamydomonas reinhardtii

32

[80]

2.4e−5

4.8e−5

–

Chlorella vulgaris

23

26

–

–

0.41B

0.44B

32

[80]

4.8e−5

2.4e−5

–

Chlorella vulgaris

22.5

27

–

–

0.44B

0.41B

32

[80]

L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate, B: Biomass Production,B: Biomass Production, μ: Specific Growth rate.

focused on the lipid content and productivity of microalgae when CO2 or organic carbon source supply energy and carbon to microalgae. It has been found that Fe augmentation with ambient or moderately high-CO2 aeration is associated with an increase in lipid content; as has been reported for C. vulgaris [78,79] and S. obliquus [71]. Ren et al. [69] discussed that the biomass and lipid production of heterotrophic microalgae exhibited a significant increasing trend with increased concentration of Fe3+, Mg2+ and Ca2+ in dark environment. EDTA addition (1.0 ×10-3 g/L) could increase the availability of iron and calcium under heterotrophic condition, thereby further enhancing the lipid production performance. However, the interactive effects of metal ions

and Chlorella sp. [65], respectively. However, the enhanced lipid accumulation under the combination of nitrogen, potassium-phosphate, and iron deprivation in case of Chlorella sp. is mainly due to the lack of nitrogen, rather than the absence of potassium-phosphate or iron [65]. It is worth noting that, the responses of microalgae to metal ions are mostly under photoautotrophic condition in which microalgae are cultured by using light as energy for photosynthesis. Heterotrophic culture, in which microalgae can utilize organic carbon to accumulate biomass and lipids in the absence of light, has been rarely investigated in the presence of metal ions. The microalgal growth and lipid production are higher under the latter condition. A few researchers have 215

a

216

↑ ~ – – – – – – –

–

– – –

– – – – – – – – –

–

– – –

Cu↑

Cu↓ Cu↓ Cu↓

–

↑

Fe↑

Cr+6↑ Cr+6↑ Ca↑d Mg↑d Ca↑d Mg↑d Pb↑ Cu↑ Pb↑

–

↑

Fe↑

– – –

–

– – – – – – – – –

–

–

–

–

–

–

C 14:1

C 14:0

↑

C 12:0

Fe↑

Fe↑

metal

– – –

–

– – – – – – – – –

–

–

–

–

C 15:0

a: C21:0: ↓↓, C24:0: ↑↑↑, b: C20:4: ↓, c: C20:4, C20:5, ↓.

Chlorella sp. Fe deprivation Chlorella v. 2.4e−5,4.8e−5 M CO2:0.036% Chlorella v. 2.4e−5,4.8e−5 M CO2:1% Chlorella v. 2.4e−5,4.8e−5 M CO2:2% Euglena gracilis Utex Euglena gracilis MAT C. vulgaris C. vulgaris S. obliquus S. obliquus C. mucigena C. mucigena C. peltigera variolosae C. peltigera variolosae C. pyrenoidosa C. protothecoides C. vulgaris

Fe

– – –

–

– – – – – – – – –

–

–

–

–

C 15:1

~↓ ↑ ↑↑

~↓

~ ↑ ↓ ↑↑ ↓↓ ↑↑ ~↓ ~↓ ~↓

↓↓

~

↑

↓

C 16:0

Table 11 The effect of metal ion on fatty acid composition of different microalgae.

~↓ ↓↓ –

~

– – – – – – ~ ~↑ ~↓

–

–

–

↑

C 16:1

– – –

–

– – – – – – – – –

–

–

–

~

C 16:2

– – –

–

– – – – – – – – –

–

–

–

~

C 16:3

– – –

–

– – – – – – – – –

–

–

–

~

C 16:4

– – –

–

– – – – – – – – –

–

–

–

~

C 17:0

– – –

–

– – – – – – – – –

–

–

–

~

C 17:1

~ ↓ ↑

~

↑ ↑ ~ ↑↑ ↓↓ ~ ~ ~↑ ~

–

–

–

↓

C 18:0

↓↓ ↑ ↓↓

~

↑ ↑ ↓↓ ↓ ↑↑ ↑↑ ↑ ↓ ~

↑↑

↓

↓

↓

C 18:1

↓ ↓↓ ↑↑

~↑

↓ ↓ ↑↑ ↓↓ ↓↓ ↓↓ ~ ~ ~↑

↑↑

↑

~

↓

C 18:2

– – –

–

↓ ↓ ↑ ↓↓ ↑↑ ↓↓ – – –

↑↑

↓

↓

↓

C 18:3

– – –

–

– – – – – – – – –

–

–

–

↓↓

C 20:0

– – –

–

– – – – – – – – –

–

–

–

↑

C 20:1

– – –

–

– – – – – – – – –

–

–

–

~

C 20:2

– – –

–

b c – – – – – – –

a

Other

~↓ ~ ↑↑

~↓

↑ ↑ ↓ ↑↑ ↓↓ ↑↑ ~↓ ~↓ ~↓

↓↓

~↑

↑↑

↑

SFA

↓↓ ↓↓ ↓

~

~ ~ ↓↓ ↑ ↑ ↑↑ ↑ ~↓ ~↓

↑↑

~↓

↓

↓

MUFA

↓ ↓↓ ↑↑

~↑

↓ ↓ ↑↑ ↓↓ ↑ ↓↓ ~ ~ ~↑

↑↑

~

↓

↓

PUFA

[75] [75] [75]

[258]

[81] [81] [72] [72] [72] [72] [258] [258] [258]

[80]

[80]

[80]

[65]

Ref

B. Sajjadi et al.

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B. Sajjadi et al.

total lipid productivity of different microalgae cells are shown in Table 12. As observed, increasing the CO2 concentration (up to ~5–7% (v/v)) significantly increases the growth rate of most algal species grown planktonically. However, higher concentration negatively affects the growth. Much research has demonstrated that Chlorella sp., Nannochloropsis oculata, Dunaliella terticlecta, C. kessleri, Spirulina sp. and C. vulgaris [87,88] had optimal growth potential in the lower range of 2–6% CO2, and the concentrations higher than that caused a decrement in the growth rate [89,90]. In contrast, the best growth potential of Chlorococcum littorate [91], Chlorella ZY-1 [92] and Scenedesmus obliquus [71], [87,93] has been observed under high CO2 level (10–15%). Aside from cell growth, enhanced lipid production in cell at various CO2 concentrations has also been reported [84,93]. For example, S. obliquus SJTU-3 culture has shown great abilities of CO2 biofixation under the high CO2 level. The total lipid productivity (25.1–95.35 mgL-1d-1) and total lipid contents (4.21–33.14%, w/dw.) in S. obliquus cultures exhibited an increasing trend with the increase in CO2 levels (0.3–12%) and produced the best growth potential at 10% CO2 [71]. As another example, Chlorella vulgaris has produced the maximum lipid productivity of 30 mg l-1 day-1 at 1.0 mM KNO3, 1.0% CO2 and 60 mmol photons m-2 s-1 at 25 °C [78]. In another study, the highest lipid productivity of 40 mg l-1 day-1 was obtained at 8% (v/v) CO2 in which higher saturated fatty acids were obtained [94]. Enhanced growth and lipid productivity of Chlorella vulgaris with CO2 concentration have also been confirmed by other researchers as well [95]. However, increasing CO2 concentration above the optimal level results in reduction of microalgae lipid content. It has been reported that the fatty acid content of Dunaliella salina decreases with the increase of the CO2 concentrations from 2% to 10% [96]. The lipid reduction for Thalassiosira weissflogii happens in CO2 range of 5–20% [97]. Generally, the most common process for cultivation of microalgae is autotrophic growth under which the cells harvest light energy and use CO2 as a carbon source. However, some microalgae are also able to use organic carbon instead of CO2 as the carbon source for heterotrophic growth. Compared to heterotrophic culture, photoheterotrophic culture uses light as a stimulant and could yield higher lipid content along with higher microalgae growth rate. In other words, light is required to use organic compounds as carbon source in photoheterotrophic metabolism. Mixotrophic culture is different from photoheterotrophic culture only in the need of CO2. Mixotrophic species can use organic carbon or sunlight, whatever they can get. In an analysis on the effect of carbon source on Chlorella minutissima, glucose is identified as the best carbon source, followed by glycerin, mannitol, and glycine to reach the maximum biomass, lipid content and lipid yield respectively [35]. Methanol has also been tested as another alternative. Faster growth and higher cell densities of Chlorella minutissima have also been observed by using methanol as alternative carbon source with daily administration of 0.005% and 0.1% (v/v) methanol [98]. The Chlorella cells can grow heterotrophically with 10 g/L glucose as the carbon source and accumulates about 280% more lipids and 45% more carbohydrates than autotrophically grown cells [99]. Heterotrophical cultivation of Chlorella sorokinian strain with high concentration of glucose as the organic carbon source yields lipid content as high as 56% (w/w) dry weight after 7 days against 19% lipids achieved in 30 days of photoautotrophic culture [41]. In case of Chlorella protothecoides, supplementation of glucose to the growth medium under nitrate limitation has been proven to raise the crude lipid content up to 55% (of dcw) compared to 15% under control condition [58]. Vazhapilly et al. [100] tested the growth of twenty different microalgae under two different carbon sources, glucose and acetate. The results showed that all microalgae except Nannochloropsis Oculata could grow under heterotrophic conditions using 5 g/l glucose. Among them, excellent growth was observed in Crypthecodinium cohnii using either glucose or acetate as carbon source. Besides, A. carterae, Phaeodactylum tricornutum, Schizochytrium aggregatum, Thraustochytrium aureum and

with carbon source highly depend on their concentration. For example, Caprio et al [80] reported negative interactive effects of high-Fe with very low or very high CO2 concentrations on lipid content. However, in either low or high Fe ions, the biomass production increased with CO2 up to 1% in their case. 5.3.2. Effect of metal ions on lipid composition The fatty acid content is clearly affected by metal ions. As observed in Table 11, the fatty acid saturation increases with iron concentration while the portion of unsaturated compounds decreases. This result has also been obtained while analyzing the effect of hexavalent chromium on the fatty acid composition of E. gracilis. Rocchetta et al. [81] found that the saturated fatty acids increased in the treated cells with the higher metal concentration. It might be due to the ability of the cells to incorporate carbon from the medium to form lipids storage rich in C14:0, C16:0 and C18:0 in spite of the significant decrease observed in PUFA content. However, the effect of iron concentration on FA composition of microalgae can be affected by the other factors as well. For example, the interactive effect of iron with low-CO2 concentration is similar to the individual effect of iron on lipid composition of Chlorella v. but at higher CO2 concentration, the intensity of iron effects moderated in favor of the synthesis of fatty acids with longer carbon chain. It is attributed to the high CO2 fixation and consumption, which might affect the enzymatic desaturation and elongation reactions in lipid synthesis [80]. Similar results have been observed for S. obliquus [3] and C. vulgaris [23,80]. Based on these studies, 2% CO2 can make a proper condition in synergetic effect with metal ions for accumulation of high amount of SFAs such as C12:0 and C16:0. 5.4. Carbon effects 5.4.1. Effect of carbon on lipid content of microalgae Generally, 50% of microalgae biomass weight is made of carbon (on a dry weight basis) which is mainly supplied from carbon dioxide. Accordingly, around 180 tons of CO2 can be disposed by generation of 100 tons of microalgae biomass, under either artificial or natural light [46]. Microalgae are efficient biological factories capable of taking zeroenergy form of carbon, synthesizing and then storing it in the form of natural oils or as a polymer of carbohydrates. It is well known that different sources and amounts of carbon have significant effects on the growth kinetics, content and composition of lipids in microalgae cells. An acceptable theory to justify the positive effect of CO2 presence is that as CO2 increases, unutilized CO2 is converted to H2CO3, which reduces the pH of the culture medium and affects the algal growth in consequence. pH value as high as 11 in high-density algae production systems is quite common where no additional CO2 is supplied. AbuRezq et al. [82] could control the pH of algae cultures in the range of 6.75–7.25 with CO2 injections, while the pH levels reached as high as pH 8.28 by day 15 in untreated cultures. However, the growth rate of some algae is inhibited in some specific concentration of CO2, which is likely due to overloading H2CO3 in the media, e.g. Chlorella and Nannochloropsis oculata at 10 > [83,84]. By contrast, algal growth can also be inhibited by the low carbon source if the CO2 level is low [85]. In term of microalgae growth, it should be mentioned that the low-nutrition condition combined with the optimized value of carbon source greatly shortens the microalgae cultivation cycle and improves the production efficiency. In an analysis on Chlorella minutissima using the optimum amount of carbon source, it has been observed that the maximum biomass productivity is obtained at the second day under low nutrition condition [35] and the sixth day under normal condition [86]. Besides, low nutrition has a negative effect on lipid productivity. On the other hand, increment of CO2 concentration under nitrogen depletion conditions increases the lipid content. Accordingly, in competition of nitrogen depletion and CO2 increment, the latter is the winner. The effect of CO2 levels on total lipid contents, biomass (dw.) and 217

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Table 12 The effect of carbon source availability on microalgae lipid, lipid productivity and growth rate. Carbon Sources

CO2 aeration%

Worst Conc.

Best Conc.

No* or Neg

Algal Species

L-LC%

H-LC%

L-LP g/Ld

H-LP g/Ld

L-GR g L-1

H-GR g L-1

day

Ref

CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2

0.03–50 0.3–12 5–15 5–70 0–12 0.04–18 0.03–15 2–15 0–16 0.5–12 0–50 ml/min 0.04–12 0.04–18 0.04–18 0.03–50 10–70 2–15 0–12 0.04–18 0.04–12 5–50

50 0.3 0 70% 0 18 15 15 0 12 0 0.04 0.04 0.04 50 70 15 0 18 0.04 50

10 12 10–15 10 12 6 3 2 8 1 50 4 6 18 5–10 10–20 2 6 0.04 4 5

> 10

– 4.2% – – – – – – – – 20% – – – – – 22.7 – – – –

– 33.14% – 38.9% – – – – – – 25% – – – – – 29.7 – – – –

– 25.1 – – – – 0.089 0.097 – – 0.004 – – – – – 0.084 – – – –

– 69.23 – – 0.22 – 0.161 0.143 – – 0.013 – – – – – 0.142 – – – –

0.82B 0.5Bcdw 1.7B 0.2B 0.3B 0.28 µ 0.21Bcdw 0.009B cdw 1.4Bcdw 0.6B – 0.34B 0.19 µ 0.19 µ 0.69B 0.77B 1xB 0.8B 0.26 µ 0.4B 1xB

1.84B 0.4Bcdw 2.3B 3.5B 1.8B 0.33 µ 1.48Bcdw 1.2Bcdw 6.8Bcdw 0.78B – 3.32B 0.26 µ 0.39 µ 1.55B 5.5B 1.3xB 3.5B 0.39 µ 3.32B 1.6xB

14 18 – 12 10 20 7 8 27 4 17 9–10 20 20 14 6 8 10 20 6–7 14.5

[89] [71] [85] [93] [87] [88] [90] [83] [94] [78] [95] [262] [88] [88] [89] [92] [84] [87] [88] [262] [263]

methanol glucose glycerin glucose glycine Mannitol sodium acetate Sodium bicarbonate glycerin glucose dextrose oxalic acid starch sucrose glycine sodium acetate glycerin

0–5% 10–17.5 g/L 33.73 g/L 32.96 g/L 41.20 g/L 33.36 g/L 74.76 g/L 92.30 g/L

0 10 – – – – – –

0.05 17.5 – – – – – –

> 0.05 > 17.5 * – – – – – –

Sc. obliquus Sc. obliquus Sc. obliquus Sc. obliquus Sc. obliquus Sc. obliquus Chlorella sp. Chlorella sp. Ch. vulgaris Ch. vulgaris Ch. vulgaris Ch. vulgaris Ch. vulgaris Ch. kessleri Ch. pyrenoidosa Chlorella ZY−1 N. oculata Spirulina sp. Spirulina sp. D. tertiolecta Chlorocuccum littorale Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima

– 6% – – – – – –

– 9.1% 10.2% 10.2% 17.2% 19.7% 17.6% 14.5%

– – – – – – – –

– – – – – – – –

0.25 µ 5.5B – – – – – –

1.45 µ 8.2B 7.5B 9B 2.44B 3.36B 0.5B 0.7B

5 10 10 10 10 10 10 10

[98] [35] [35] [35] [35] [35] [35] [35]

9–25 g/L 10 g/L 1 g/l 1.46 g/l 0.88 g/l 0.93 g/l 1.22 g/l 1.33 g/l 1 g/l

25 – BM BM BM BM BM BM BM

9 – – – – – – – –

– – – – – – – – –

Ch. minutissima Ch.protothecoides Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima Ch. minutissima

0.7 g/l 14.6b 1x 1x 1x 1x 1x 1x 1x

0.75 g/l 55%a 3.1x 3.33x 2.8x 4x 3.7x 2.6x 5.7x

– – – – – – – – –

– – – – – – – – –

0.2B – 1xB 1xB 1xB 1xB 1xB 1xB 1xB

0.55B – 2.7xB 1xB 1.1xB 1.13xB 2.15xB 1.99xB 3.6xB

15 – 7 7 7 7 7 7 7

[86] [99] [264] [264] [264] [264] [264] [264] [264]

< 15% < 10 < – < > < – < – – – > < > –

3< 2 8< 50

10 10, > 20 2

>5

a: heterotrophical, b: photoautotrophic, c: mixotrophic culture, B: Biomass Production, BM: Basic Medium [264], CDW: Cell Dry weight, L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate, B: Biomass Production, μ: Specific Growth rate, Worst and best concentrations in terms of Lipid content and Lipid Productivity. Ch. vulgaris: Chlorella vulgaris, Ch. minutissima Chlorella minutissima, Ch. protothecoides: Chlorella. Protothecoides, Ch. Kessleri: Chlorella kessleri, Ch. Pyrenoidosa: Chlorella pyrenoidosa, N. oculata: Nannochloropsis oculata, N. sp: Nannochloropsis sp., D. tertiolecta: Dunaliella tertiolecta.

concentration of carbon dioxide (2–10%) induces the accumulation of saturated fatty acids. This observation has been confirmed by many researchers [104]. However, production of saturated fatty acids outpaces the unsaturated compounds at very high CO2 concentration (> 10%). In other words, it can be stated that, the degree of unsaturation is reduced by increasing the CO2 concentration.

Amphidinium sp. demonstrated very high growth using glucose. The last three showed moderate growth under acetate (1 g/L). However, A. carterae, Chröomonas salin, Cryptomonas sp. Nannochloropsis oculata, Pavlova lutheri, P. lutheri, Por. Purpureum, Prorocentrum minimum could not use acetate as the carbon source and grow [100]. Some authors have suggested that Sodium bicarbonate and HCl do not only control pH but also form carbonic acid, which can be broken down into CO2 and water [101]. Many microalgae can also directly use Bicarbonate as for their carbon source for photosynthesis [102,103].

5.5. Salinity effects 5.5.1. Effect of salinity on lipid content of microalgae Salinity is the other environmental factor which not only affects the growth and productivity of algae but also limits contaminants and competing microorganisms. High salinity causes high extracellular osmotic pressure, resulting in stress generation inside the alga cell, which is reacted in physiological and biochemical mechanisms. Restoration of turgor pressure, regulation of the uptake/export of ions through the cell membrane, and accumulation of osmo-protecting solutes and stress proteins become active when microalgae cells are exposed to salinity. High salinity stress mainly affects membrane fluidity and permeability [35]. In very high concentration, salinity can damage the microalgae

5.4.2. Effect of carbon on lipid composition The lipid profile of the microalgae has been reported in a few studies as summarized in Table 13. It is observed that by increasing carbon monoxide, the portion of C18:1 and C18:2 increases in all compounds while the content of C18:3 dramatically decreases. However, the intensity of this increment or reduction is strain-specific. Accordingly, it is difficult to judge the effects of CO2 on the total amount of saturated and unsaturated compounds. However, as a general idea, it has been reported that very low concentration of CO2 (< 2%) facilitates the production of unsaturated fatty acids (C18:1, C18:2) whereas high 218

219

[267] [263]

[89]

↑↓ 10 ↓↑ ↓ ↑↓ 10 ↑↓ ~ – – – –

– – –

– –

b c

↓↑ 10 ↑↓ ↓

↑ ↓ ↑ ↑ ↑ ~ ↓ ~ ↓ – – a – – – – ~ ↓↓ – ~

↓ ↓↓ ↓↑ 10 ↓↑ 10 ↓↑ ↑ ↑↓ – – – – ↓↓ ~

~ ↑

1–10 0–8 0.03–50

0.03–50

1–53 μM/l 5–50

Ch. pyrenoidosa

Emiliania huxleyi Chlorocuccum littorale

– –

– –

– –

↓↑ 10 ↑↓ ↓ – – – ~

0.03–2

Ch. vulgris Air-Air CO2 riched Ch. vulgris Ch. vulgris S. obliquus

a: C20:5, ↑, b: C18:4 ↓↑, C18:5 ↓, C22:6 ↓, c: all compounds have not been analyzed, < 10%.

– –

–

↓↑

~

↑↓ 10 ↑ ~

↑↑ ↑ ↑↓ 5–10 ↑↓ 10 ↑ ↓ ↑ ↑ ~ ↓↓ ↓↓ ~ ↑ – – – – –

– – ↓↑ 10 ↑

– – ↑↓ 10 ↑↓ 10–20 – – ~↑ – ~ ↑↑ ↑ ↓ – – – – – – – ~ ~

↓ – –

[265] ↓ ↑ ~↑ – – – – ↓↓ ↑↑ ↑↑ ~↑ – – ↓ ↑ ~ ~↑ – – ~

–

C 16:1 C 16:0 C 15:1 C 15:0 C 14:1 C 14:0 CO2 Edible Oil

Table 13 The effect of carbon availability on fatty acid composition of different microalgae.

C 16:2

C 16:3

C 16:4

C 17:0

C 18:0

C 18:1

C 18:2

C 18:3

C 20:0

C 20:1

C 20:2

other

SFA

MUFA

PUFA

Ref

cell, but an optimal stress can increase lipid production. Various sodium salts (NaCl, Na2S2O3, NaHCO3, C2H3NaO2) have been used to induce lipid accumulation of different microalgae (in two phase mode) and NaCl has been found to increase the lipid content dramatically while demonstrating a slight reduction in biomass production [44]. In order to cope with the contradiction between lipid production and biomass production yield, a culture mode that allows an optimum growth rate and permits a lipid enhancement was recommended by some researchers [105]. Toward this objective, optimal conditions (free or moderate concentration of salt) to highly concentrate biomass production are provided in the first phase, while stressful conditions are employed to induce lipid biosynthesis in the second phase. Most microalgae regulate lipid biosynthesis as a physiological resistance strategy to salt stress. However, salinity tolerance capability of every strain is different. Table 14 lists the effect of salt stress on lipid content, growth rate and lipid productivity of many microalgae. As per Table 14, C. nivalis has been found to be extremely resistant to NaCl stress and can survive under the lethal NaCl concentration of 70.13 g/L (1.2 M) [106]. Dunaliella sp. is also one of the most resistant microalgae to high salt concentration. Their ability to get benefit from salinity stress in order to increase the biomass growth and lipid content makes them one of the most suitable strains to analyze the effect of salinity. It has been reported that with an initial NaCl concentration of 1.0 M, an addition of 0.5 or 1.0 M NaCl at mid-log phase or the end of log phase during cultivation, Dunaliella can reach the lipid content of 70% but at the cost of inhibited cell growth [35]. As per Table 14, higher lipid contents are observed in most microalgae that are subjected to salt stress e.g. Scenedesmus species [85,107] Chlorella. vulgaris [108], Chlamydomonas Mexicana, [109], Scenedesmus sp. [110]. In the case of Dunaliella tertiolecta, salinity increment (0.5 M) does not only increase the lipid content from 60% to 68% but also increase the TG in lipid by 17% [111]. This concentration of salt is also found in favor of the total carotenoid production in Dunaliella tertiolecta [112]. Low concentration of salt (0.43 mM) induces carotenoid accumulation in Haematococcus too, while high levels of salinity is lethal for it [113]. On the other sides, Chlorella minutissima reluctantly grow in the medium with 20 g/L NaCl and its growth is inhibited under higher NaCl concentration. Generally, most microalgae reach the maximum lipid content at an optimum salt concentration after which the lipid content sharply decreases. This concentration has been reported to be between 20 and 40 g/L (0.34–0.68 mol/L) in most microalgae. For examples, lipid accumulation and biomass synthesis of Chlorella minutissima [35] and Nitzschia Laevis (Bacillariophyceae) [114] under salinity stress till an optimum values of 40 and 20 g L-1 respectively, have significantly increased while negative effect has been observed by further increasing salinity. In case of Nitzschia laevis, NaCl optimum concentration is about 10 g L-1 beyond which polar lipids increase but storage lipid (i.e. TAG) decrease [114]. In some cases, elevated salinity conditions beyond the optimal value leads to increased storage lipid content and decrease in membrane lipid content [115]. These variations in lipid and fatty acids confirm a decrease in membrane fluidity and permeability under salinity stress, which can help the algae bear and acclimate to these conditions. Increment of protein and carbohydrate by salinity have been observed in other microalgae i.e Ankistrodesmus falcatus [116]. Some microalgae produce carbohydrates with low molecular weight in response to salt stresses, mainly in cyanobacteria [104,117–120]. Increment of carbohydrate and lipid simultaneous with reduction of protein due to salinity stress was also reported in case of Scenedesmus sp. [110]. Some micro algae are also less sensitive to salinity [35]. Different authors have reported that salinity stress does not have any effect on lipid and carbohydrate content of Botryococcus braunii alga strain [68,70,120], but protein content decreases [121]. Ben-Amotz et al. [122] found similar results in terms of protein and carbohydrate of Botryococcus braunii but higher lipid accumulation under the salinity of 0.5 M. Rao et al. [120] reported that salt concentration of 34 mM induces significant higher biomass and carbohydrate and carotenoids

[266] [94] [89]

Renewable and Sustainable Energy Reviews 97 (2018) 200–232

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Renewable and Sustainable Energy Reviews 97 (2018) 200–232

B. Sajjadi et al.

Table 14 The effect of salinity on microalgae lipid, lipid productivity and growth rate. Salinity M

Worst Conc.

Best Conc.

Negative effect

Algal Species

0–1.4 M 0.5,1 M 0.1,0.2,0.3 MI 0–0.1 M 0–0.001 M 0–0.4 M 0.2–1 M 0–1.2 M 0–0.7bM 0–0.5dM NaCl 0–0.3 M NaHCO3 0.04–0.34 M 0–0.7b M 0–0.7b M 0–0.1 M 0–0.34 M 0–0.085 M

1.4 0.5 0.1 0 0.0002 0 0.2–0.4 0 0.17 0d

0.7 1 0.3 0.025 0.001 0.4 0.6 0.6 0 0.34d

< – – < < < < < < <

Ch. minutissima Du. tertiolecta Sc. obliquus Sc. obliquus Sc. quadricauda Sc. sp. N. salina N. oculata N. oculata De. abundans

0

0.3

–

0.04 0 0.17 0 0 0

0.17 0.17 0 0.025 0.34 0.02–0.034

0–0.34 M

0.34

0, 0.08 0.17 0.1 0.17

< < < < < < > <

I

0.017,0.034,0.5 M 0–0.14 M 0–0.7bM

0.5 0 0.7

0.34–0.7 <

0.025 < 1 0.4 0.6 < 0.6 0 0.34 <

0.17 < 0.17 < 0 0.025 < 0.34 0.017, 0.034 0.08

> 0.17 – < 0.17 <

L-B B:g/l

H-B B:g/l

day

Ref

0.324gld – 0.058

8B 1B – 0.25B 1.5B 0.4B dcw 1xB 2.15B – 0.19BP

7.5B 1.03B – 0.65B 1.35B 0.12B dcw 7xB 1.91B 0.5aB 0.16BP

4D+3 10I 15 20I 15I 15I 35I − 40I 5D + 4 14a+ 3b 8c+ 6d

[35] [111] [85] [123] [107] [110] [115] [105] [268] [44]

0.045

0.054

0.19BP

0.16BP

8c+ 6d

[44]

– – –

– – –

0.051 –

0.061 –

– – – < 0.1B 0.15BP 0.9B

–

–

0.8B

20 10a+ 1b 10 a+ 2b 20I 12c+ 8d 18 18 20

[116] [268] [268] [123] [269] [120]

31, 22 – 53.93% 47

– – –

– – –

– 0.25aB 0.28aB 0.8B 0.13BP 1.5B 1.35B 0.9 B 1.1B 5.67B 5.47B 0.8aB

– 5 12a+ 2b

[114] [108] [268]

L-LC %

H-LC %

G G G G G G G G G G

15% 60.6% 15% 18 6.2% dcw 18.98 < 15% 35 17 23

31.82% 67.8% 30% 34 6.9% dcw 33.1% 36% 44.5 29 35.5

De. abundans

G

23

An. falcatus Du. salina Du. tertiolecta Chl. mexicana Sc. obtusus Bo. braunii

G G G G G PC

Bo. braunii

PC

Nitzschia laevis Ch. vulgaris Is. galbana

b

GB YG B

L-LP gld

H-LP gld

– –

– –

– –

– –

0.231gld – 0.045

33.44

43 22 23 15 35.3 –

56.1 43 40 37 47.7 –

26 – 47.7% 30

B

10.1 5.47B –

[68]

L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate, B: Biomass Production, Worst and best concentrations versus Lipid content and Lipid Productivity, D: Initial Days without salt (2 phase grwoth), I: initial salt, *: 2phase culture, a: initial condition salt:30psu (0.5 M)b: second phase, d: second phase PC: Phylum: Chlorophyta, BP: g/lday, c/d: phase I(c) without salt, Phase II(d) different salt concentration. De. abundans: Desmodesmus abundans, Is. galbana: Isochrysis galbana, An. falcatus: Ankistrodesmus falcatus, Chl. Mexicana: Chlamydomonas Mexicana.

in the formation of very long chain fatty acid derivatives through chain elongation [77]. However, it should be noted that, this trend was consistent till the optimum concentration of salt or the concentration with which the strain could adapt itself and after that the trends sometime changed. For example, Cao et al [35] reported that the degree of fatty acid unsaturation in both polar and neutral lipid fractions increased sharply till the optimum concentration of NaCl, but decreased at higher NaCl concentration.

production in Botryococcus braunii compared to the control conditions. Accordingly, it can be concluded that salinity contributes to different effects (i.e. levels of storage type or content) for different algae species.

5.5.2. Effect of salinity on lipid composition of microalgae Environmental salinity as any other physiological state of the culture affects the fatty acid profile of the intracellular lipid of microalgae (Table 15). The fatty acid profile of the intercellular lipids confirms that the relative content of monounsaturated FAs remarkably (i.e. palmitic (C16:1) and oleic acids (C18:1)) increases under high salinity and high alkalinity compared to the control without any salt addition. These compounds mainly make up the neutral lipids which in turn favors biodiesel production [44]. On the other side, the proportion of polyunsaturated FAs (PUFA), especially linolenic acid (C18:3), decreases substantially. These changes are in the direction to keep the membrane fluid and prevent its destruction. These results are consistent in the cases of Chlamydomonas Mexicana [123], Scenedesmus obliquus [123], Desmodesmus abundans [44], Dunaliella sp [124], Botryococcus braunii [120], Cladophora Vagabunda [125]. In term of saturation, some authors have reported that the fatty acids of membrane lipids are desaturated, leading to increment in the proportion of unsaturated fatty acids, e. g. as in Boekelovia hooglandii [126]. The same trend has also been observed in the fatty acid composition of polar lipid in Dunaliella salina Teodoresco by the change in NaCl concentration [127]. However, increment in the saturated fatty acids under high-salt stress has been reported by a few cases of Dunaliella sp and Nitzschia laevis [114,124]. In the case of Botryococcus braunii, the saturated compounds have not decreased significantly but they change remarkably. In the intercellular of this microalga, C18:0 reduces significantly while C16:0 increases. There is also a remarkable content of C22:0 and C24:0, which are not generated under control condition [120]. In B. braunii, the fatty acids with 18 carbon atoms in their chain have been reported as the precursor

5.6. pH effects 5.6.1. Effect of pH on lipid content of microalgae Acidity or basicity of culture medium is another factor that directly affects the microalga growth rate and species composition. The pH value affects the availability and absorption of nutrients such as iron and carbon, which indirectly affects the growth rate [104]. pH in microalga cultures rises steadily during the day due to photosynthesis and the use of CO2. During the night (dark), respiration reverses this process and lowers pH levels again. pH is normally controlled by injection of CO2 (also used as carbon source) into the culture, using buffer or injecting mineral acids into the culture medium [128]. pH value may reach 10 when no CO2 is supplied and the values of 11 or above is not uncommon if CO2 is limiting and bicarbonate is used as a carbon source. This range of pH is quite inappropriate for most microalgae. It has been reported that the pH of Nannochloropsis, Tetraselmis, and Isochrysis can be maintained within the range of 6.75–7.25 with CO2 injections, while untreated cultures reached the pH of 8.25 within 15 days [82]. In recent studies addition of HCl and Sodium bicarbonate has also been reported to control pH as in Tetraselmis suecica and Chlorella sp cultural medium, [101]. These compounds can form carbonic acid followed by decomposing into CO2 and water consequently. Many microalgae also use bicarbonate as an inorganic carbon source for photosynthesis [129]. 220

221 ↑↓ 0.7 M

↑

↑

Nannochloropsis sp.

~

~

↑

–

↓

–

↓↑ 25 ~

↓

– ~

–

–

–

–

~ – –

↑ ↓ ↑↓↓ ↑ ↑

C 16: 2

C 16: 1

~

Nannochloropsis sp.

–

–

–

–

↓

↑↓ ↑↑ ↓

C 16: 0

↓ ↓ ↓

–

↓↑ ~ ~

Boekelovia hooglandii Cladophora Vagabunda Nannochloropsis sp.

–

–

–

–

– –

C 15: 1

↓ ~ ↓

–

–

Scenedesmus obliquus

–

–

–

–

Dunaliella salina polar lipids Chlamydomonas mexicana

–

~ – –

C 15:0

–

–

–

–

–

– –

C 16: 3

–

–

–

–

–

↓ – –

C 16: 4

–

~

– ↑ ~

~

~

–

↑

~ ↓↓ ↑

C 18: 0

↑↑

↓

↑↓ 25 ↑↓ 25 ↑ ↑↑ ↓

↓

↑↑

↑ ~ ↑↑ ↑↑

C 18:1

~

↑

↓↑ 25 ↑↓ 25 – ↑ ↑

↑

↓

↑ ~ ↓↓ ↓

C 18:2

↓↑ 25 ↓↑ 50 – ↓

↓

↓

↓↑ ~ – ↓

C 18: 3

~

–

–

–

~

– ~

C 20:0

↓

↑

↑

–

–

–

–

–

– –

C 20: 1

–

–

–

–

–

–

–

– –

C 20: 2

k

j

g i j

–

–

f

e

a c,de

other

↑

↓

↓ ~↓ ↓↓

~

↓↑

↓

↓

↑ ~ ~↓ ↓

SFA

↑↑

↓↓

↓ ↑↑ ↓↓

↑↓

↑↓

↓↑

↑↑

↑ ↓ ↑↓ ↑↑

MUFA

↓↓

↑↑

↑ ↓ ↑↑

↓

↓↑

↑

↓

↓ ↑ ↓ ↓↓

PUFA

[270]

[270]

[126] [125] [270]

[123]

[123]

[127]

[44]

[124] [114] [120] [44]

Ref

TAG: Fatty acid profile of TAG, A: C20:5: ↑. B: C13:0↑↑, C: C22:1 ↓↓ D: C24:0 ↑↑ E: C22:0, ↑, e: C22:0 ↓, f: C14:2 ↑↑, g: C18:4, ↑, C20:5 ↑↓, C22:5↑↑, C22:6↑↑, i: C18:4 ↓, C20:4 ↓, C20:5 ↓, C22:5 ↓, C22:6 ↓, j: C20:4 ↑, C20:5↑↑, k: C20:4 ↓, C20:5↓↓.

–

25

0–100 mM

0–0.8 M 50%− 200% NaNO3 150–3000 µM NaH2PO4, 6–120 µM NaCl 0.2–1 M

25

0–100 mM

–

↓

Desmodesmus abundans

25

C 14: 1

~ ~ – –

C 14: 0

~ ↓ – ↓

Dunaliella sp Nitzschia laevisTAG Botryococcus braunii Desmodesmus abundans

10 85 20

0.4–4 M ↑10,20,30 g/l 0–85 mM 0, 20 g/l NaCl 0, 25 g/l NaHCO3 ↑0.85–3.4

Optimum salt Concentration

best

Edible Oil

Table 15 The effect of salinity on fatty acid composition of different microalgae.

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Renewable and Sustainable Energy Reviews 97 (2018) 200–232

222

9 – – – 7.2 7.2 7.2 10 10 5.5

3 &12 pH: 7.5 9.5 9.5 pH: 7.5 – 6.5

5,10 8

4–11 5–8 5–11c < 10- > 11 7.2,9.5 7.2,9.5 7.2,9.5 7.6,10 7.6,10 5.5–8

3–12c 7.5–11.1 7.5, 8.5, 9.5 7.5, 9.5 6.5–10 7–9 6.5–8

5–10 4–8

8 4–6

6–11 8.4–11.1 7.5 7.5 9.7–10 8 7.5

6–7 7 7 < 10–11 9.5 9.5 9.5 7.6 7.6 7

Best pH

>8 <7 < 7, > 7 > 11 – – – – – <7 >7 < 6, > 11 – – – – 7&9 < 7.5 > 7.5 < 8, > 10 >6

Negative or No effect

Pavlova lutheri Nannochloropsis sp.

Sc. sp R− 16 Sc. sp w Sc. obliquus Ch. vulgaris Coelastrella sp. p Nannochloropsis Tetraselmis suecica

Ch. minutissima Chlorella sp. Chlorella sp. Chlorella. Chlorella FGP5 Chlorella Rb1a Chlorella RBD8 Chlorella OS1–3 Chlorella OS4–2 Chlorella

Algal Species

24%, 23% 25%

17% 0.6%b – – 0.1%b 17% –

1% – 15% – 10% 14% 5% 37% 15% –

L-LC

35% 37%

42 > % 16.8%b – – 0.6%b 25% –

14% – 33% – 20% 17% 23% 44% 36% –

H-LC

– – – – – 0.09 gld – –

– –

– – – 171a g/dw – – – – – 90mgld

– – – 103a g/dw – – – – – 10mgld

– – – – – 0.030 gld

H-LP

L-LP

– –

– – – – – 400 mgl

– 0.11gl 50 mg L−1 – – – – – – 200mgl

L-BY gL-1

– –

– – – – – 950 mgl

– 0.15gl 167.5 mg L−1 – – – – – – 1600mgl

H-BY gL-1

0.4 –

B

3.3B 0.83B 1.6B 1.35B 0.71B – 0.3 g/ld

0.7B 0.97B 0.95B 0.7B 0.86B – 0.1 g/ld B

1.71 g/ld 0.4 g/ld

1.62 g/ld 0.1 g/ld

0.2 –

8.5 1.3 B 0.47B – 0.382 g/ld

B

GR-B 1.2 0.85 B 0.39B – 1.175 g/ld

B

GR-W

~6 –

6 28 6 6 14 21 50

10 15 30 10 7 7 7 7 7 50

day

[273] [274]

[271] [135] [272] [272] [135] [129] [101]

[35] [137] [138] [140] [139] [139] [139] [139] [139] [101]

Ref

L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, BY: Biomass Yield GR: Growth Rate, B: Biomass Production and rest: Specific Growth rate, Worst and best conditions in terms of Lipid content and Lipid Productivity, a: μmol g-1dry wt, b: % TAG, c: initial pH, D: growth experiment within 3 days.

Worst pH

pH

Table 16 The effect of culture pH on microalgae lipid, lipid productivity and growth rate.

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saturated fatty acids in membrane lipids of microalgae at under pH stresses (a pH value higher or lower than their originality) represents an adaptive reaction to decrease membrane lipid fluidity [141]. Therefore, changes of pH do not induce any stress unless the change is opposite of the originality of microalgae. For example, in the case of Nannochloropsis, the maximum lipid content has been obtained at pH 8 while accumulation of unsaturated, saturated, and mono/polyunsaturated lipid does not change by reducing or increasing the pH value to 7 and 9 respectively [129]. Skrupski et al [139] also reported that, the saturated and polyunsaturated content of both phospholipid and TAG increased while the monounsaturated decreased at high pH values. However, high pH values do not mean pH stress for all Chlorella strains since some of them originate from alkali medium culture [139].

Although different species might have different growth responses to the changes of pH values, the acceptable pH range for most microalgae species is 7–9. However, the optimal pH range for microalgae growth is strain-specific and narrow. It is between 8.2 and 8.7 for most of them [104]. For example, the optimal productivity of cyanobacterium Anabaena variabilis is in a medium with pH range of 8.2–8.4, being slightly lower at 7.4–7.8. The productivity decreases dramatically above pH 9, and the cells are unable to thrive at pH 9.7–9.9 [130]. The maximum growth rate of Nannochloropsis is also observed in pH range of 8–9. It is significantly lower at nutrient medium culture (pH 7) and it is stopped at pH lower than 6 and higher than 10 [129]. The pH value of 8 for keeping the optimal growth rate of Nannochloropsis has also been confirmed in other work [131]. Consistent decline of growth rate and photosynthesis of Thalassiosira pseudonana and Thalassiosira oceanica has also been reported at high pH levels (> pH 8.8) [132]. By analyzing the growth rate of Skeletonema costatum under different pH conditions ranging from 6.5 to 9.4, the maximum growth rate was observed at pH 7.5 while it significantly reduced at pH > 8.5 [133]. The maximum biomass and lipid productivity of T. suecica have been reported to be in pH range of 7–7.5 [101]. However, some species are acclimated to high alkali or high acidic environments. As reported, the cells of Chlorella protothecoides grow rapidly at the pH value of 5.0, (compared to the pH of 4.0, 4.5, 5.5, 6.0, 6.5, 7.0) and its growth is inhibited under alkaline pH [134]. In contrast, Scenedesmus sp [135] grow better under alkali conditions (pH > 9) and have higher lipid productivity in these conditions. Generally, high alkali conditions seriously inhibit the normal growth of most microalgae while acidic conditions are conducive for microalgae growth and oil accumulation [35,114]. It is known that high level of pH is often associated with harmful microalgae blooms or red tides [135,136]. Lipid content is also dramatically affected by pH value. As per Table 16, the pH value ranging from 7 to 9.5 can also yield a higher lipid accumulation in most microalgae species. Under nitrogen limitation, Chlorella could yield higher triacylglycerol (TAG) production with a rise in the culture medium pH [135]. Rai et al [137] also showed that the lipid content of Chlorella sp. increased to 23% at pH 8 though the highest biomass production was observed at lower pH (=7). The same trend was exactly reported by Zhang et al [138]. The authors observed the maximum algal lipid yield at initial pH of 7.0. The triacylglycerols content was significantly enhanced to 63.0% at initial pH of 5.0 and biomass accumulation was at its maximum level at pH 9. Based on that work, although the lipid content dramatically changed by pH, the total TAG and biomass production did not change significantly at pH range of 5–9. Skrupski et al [139] analyzed five different strains belonging to the genus Chlorella but three of them (FGP5, Rb1a, RBD8) originated from a medium culture with pH 7.2 and two of them (OS1-3 OS4-2) from an environment with pH 10. Accordingly, the researchers considered the pH values of 9.5 and 7.6 in order to put the microalgae under stress and found the lipid increment in both groups. However, in the first group, lipid increment came at the cost of lower mass productivity but in the second, mass productivity increased.

5.7. Temperature effects 5.7.1. Effect of temperature on lipid content of microalgae Seasonal temperature fluctuations, daily temperature variations, and abrupt temperature variations due to any reason can modify the growth conditions of microalgae and thus its production efficiency or vice versa. Generally, outdoor photo-bioreactors induce the temperature fluctuations of 10 and 45 °C (depends on the region) the microalgae. Inconsistent with this assumption that the lipid metabolic pathways of microalgae may change in a way resulting in higher lipid accumulation and higher ability to resist external temperature stress, both low and high temperatures may conceal lipid accumulation. Moreover, such situation severely inhibits microalgae growth [142]. Among them, the effect of elevated temperatures is more deleterious than low temperatures. In the case of C. vulgaris, if the temperature reaches 38 °C, an abrupt interruption happens and later the cells die. These changes are easily visible because of the change in cells color from green to brown, and the growth rate, which divert to negative values [46]. Though most microalgae species are capable of carrying out cellular division and photosynthesis over a wide range of temperature (15 and 30 °C), the temperature optimal range differs for various microalgae. It is about 32 °C for Dunaliella sp. [143], 25–30 °C for of C. Vulgaris [46] and 15–20 °C for Chlorella Minutissima [35]. Generally, Chlorella species have the optimal growth rate over a wide range of temperatures. Studies of 17 different Chlorella strains have demonstrated that this group of microalgae can grow successfully between 26 (i.e. C. Prothotecoides, C. Vulgaris) and 36 °C (i.e C. Kessleri, C. Fusca,) [144]. Accordingly, microalgae are divided into three categories based on their optimal growth rate at different temperatures, which are 20 and 25 °C for mesophilic species; 17 °C for psychrophilic strains (Asterionella Formosa) or increase to 40 °C for thermophilic strains (Anacystis Nidulans, Chaetoceros). Microalgal growth under above-optimal temperature is speciesdependent and except for the thermophilic species, it has been rarely reported for mesophilic and psychrophilic strains. Moreover, the optimal growth temperature may change slightly by the medium culture condition. For example, the optimal growth temperature of Dunaliella tertiolecta increase by 6 °C by increasing the sodium chloride content from 0.125 to 1.5 M [145]. It should be noted that, a balance must be maintained between the energy consumption inside the cell and the photosynthetic energy supply in order to grow microalgae. An imbalance between energy supply and consumption, caused by environmental factors results in a change of the photosynthetic apparatus (Rubisco activity, unit size). Some microalgae shrink their cells in order to cope with the imbalance between anabolic and catabolic processes due to temperature rise. In other words, microalgae reduce their volume in order to reduce metabolic costs and enhance uptake rates [146]. Such acclimation has been observed in the cases of Microcystis aeruginosa, Scenedesmus acutus and Dunaliella species by reduction of photosynthesis rates, respiration rates and cell size [147,148]. Aside from rapid physiological adjustments, slow generational adaptation is another solution in response to

5.6.2. Effect of pH on lipid composition of microalgae pH variation in the culture medium also affects the lipid composition accumulated in the microalgae, as summarized in Table 17. Generally, alkali pH stress results in generation of lipid with higher saturated compounds in most microalgae originated from acidic medium. For example, alkaline pH stress significantly reduces the glycolipid and polar lipid content of Chlorella while increasing the TAG accumulation, which is not dependent on nitrogen or carbon limitation levels. However, lower total lipid with higher amount of saturated (C16:0) and monounsaturated (C18:1) is the resultant outcome [140]. Consistent with this, Analysis of pH effect on an unidentified strain (Chlamydomonas sp.) originated from an acidic lake shows that FAs of polar lipids are more saturated compared with that microalga (C. reinhardtii.) isolated from a medium with higher pH. In other words, the increase in 223

pH

Edible Vegetable Oil

224

a: Phospholipid, b: TAG.

< 10 -> 11 ↑7–9 ↑7.2,9.5 ↑7.2,9.5 ↑7.6,10 ↑7.6,10 7.2, 7.2–9.5 7.2–9.5, 9.5 10, 7.6–10 7.6–10, 7.6 8.1–10↑

< 10 -> 11 ↑7–9 ↑7.2,9.5 ↑7.2,9.5 ↑7.6,10 ↑7.6,10 7.2, 7.2–9.5 7.2–9.5, 9.5 10, 7.6–10 7.6–10, 7.6 8.1–10↑

Chlorella. Nannochloropsis Chlorella FGP5a Chlorella FGP5b Chlorella OS4–2a Chlorella OS4–2b Chlorella FGP5a Chlorella FGP5b Chlorella OS4–2a Chlorella OS4–2b N. oleoabundans

Chlorella. Nannochloropsis Chlorella FGP5a Chlorella FGP5b Chlorella OS4–2a Chlorella OS4–2b Chlorella FGP5a Chlorella FGP5b Chlorella OS4–2a Chlorella OS4–2b N. oleoabundans

pH

Edible Vegetable Oil

↓ ~ ~ ↑ ~ ~ ~ ↑ ~ ~ ↑

C 18:0

~ ~ ~ ~ ~ ~

C 14:0

↑ ~↑ ↓ ↓↓ ↑ ↑ ↓↓ ↓↓ ↑ ↑ ↑ ~ ~ ~ ↑ ↓ ↓↓ ↑↑ ↑↑ ↓ ↓ ↑

C 18:2

– ~

– –

C 18:1

C 15:0

C 14:1

Table 17 The effect of pH on fatty acid composition of different microalgae.

↓ ~ ↑ ↑↑ ~ ~ ↑ ↑↑ ↓ ~ ↓

C 18:3

– –

C 15:1

– –

C 20:0

↑ ~ ↑ ↑ ↓ ~ ↑ ↓↓ ~ ~↓ ↑

C 16:0

– –

C 20:1

~ ~ ↑ ↓↓ ~ ~ ↓↓ ↓↓ ~ ~ ~↑

C 16:1

– –

C 20:2

~ – ↓ ↑ ~↓ ~ ↓↓ ↑ ~↑ ~

C 16:2

↑ ~ ~↑ ↑ ~↓ ~ ↑ ↓ ~ ~↓ ↑

SFA

~ – ~ ↑ ↑ ~ ↑ ↑ ↑ ~

C 16:3

↑ ~ ~ ↓↓ ↑ ↑ ↓↓ ↓↓ ↑ ↑ ↑

MUFA

~ – ~ ~ ~ ~

C 16:4

↓ ~ ~↓ ↑ ↑ ↓↓ ~ ↑↑ ↓ ↓ ↑↑

[140] [129] [139] [139] [139] [139] [139] [139] [139] [139] [275]

Ref

– – –

– – –

PUFA

– –

C 17:1

– ~

C 17:0

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tertiolecta [154] and Dunaliella salina [155] have also demonstrated similar acclimation. In the case of Skeletonema costatum, the response is different and the chlorophyll content increases with lower temperature. Arrhenius equation is the most popular model to explain the relation between below-optimal temperatures and growth rate. According to this model, cell division, growth and photosynthesis in most microalgae are expected to double for each 10 °C increase until unfavorable temperature is achieved. In other words, the temperature coefficient Q10 is expected to present a value near 2 by the Arrhenius model. Microalgae growth rate significantly decreases as temperature exceeds the optimal temperature, due to the unfavorable effect of heat stress on the functionalities of enzymes and proteins. The response of microalgae growth to the temperature enhancement is often described by a bell-shaped growth curve. The temperature in which the growth rate reaches zero value is named as lethal temperature, which is from 30 to 35 °C onwards for mesophilic microalgae [156,157].

temperature elevation, which is the case for Scenedesmus. It has been observed that this microalga could adapt to 30 °C after 15 generations, to 35 °C after 30 generations and to a maximum of 40 °C after 135 generations [149]. Converti et al. [46] found that increasing the temperature from 20 to 25 °C doubled the lipid content of Nannochloropsis oculata (from 7.9% to 14.9%) at the cost of reduced growth rate. In some cases (i.e. Isochrysis galbana [150]), high growth temperature has been associated with decreases in carbohydrate and lipid and increase in protein content. In case of Isochrysis galbana, lipid classes and fatty acid profiles of the cells changed with the specific growth rate of the culture. Neutral lipids decreased while glycol and phospholipids increased with the dilution rate [150]. The effect of temperature fluctuation, especially reduction, on total lipid content of microalgae has been widely studied, as summarized in Table 18. The temperature effects on lipid yield vary depending on different species. As observed, in most strains, low temperature causes stress in favor of higher lipid accumulation. It has been found that low temperature stress has slight effects on carbohydrate production while reducing carbon incorporation into proteins. A possible conception to justify this observation is that the biosynthesis of amino acid and putative osmolyte biosynthesis is inhibited as the temperate reduces. Therefore, glycolysis intermediates, which can play as the role of precursors of acetyl-CoA increases, leading to the synthesis of relatively more fatty acids [151]. A decrease in cultivation temperature from 25 to 20 °C increases the lipid content of Scenedesmus sp by 2.5 folds with only a slight effect on the growth rate (8% loss) [152]. The same trend has been observed in the case of Chlorella vulgaris, for which a temperature drop from 30 to 25 °C increases the lipid content by 2.5 folds without any effects on the growth rate [46]. However, in some cases i.e. Chlorella sorokiniana, although low temperature (18 °C) increases the lipid content, algal growth is inhibited at 18 °C and so the lipid productivity only reaches 76% of that at 26 °C [151]. In order to alleviate the adverse effects of low suboptimal temperatures on lipid productivity, Wang et al. [151] added Glycine betaine (GBT), a quaternary ammonium compound, to the media at the initial phase of culturing. The authors reported that GBT could enhance the biomass, lipid content and lipid productivity of C. sorokiniana by enhancing carbon fixation at both the optimal temperature of 26 °C and a low sub-optimal temperature of 18 °C without any effects on its composition. Moreover, low temperatures generally decrease the carboxylase activity and so the energy supply is overproduced if the light condition does not change, creating light saturation conditions. Chlorella vulgaris can manage this imbalance and grow successfully at 5 °C with low chlorophyll content [153]. Dunalliella

5.7.2. Effect of temperature on lipid content of microalgae Although in most research, the focus is on total lipid, some researchers have also examined the effect of temperature on different classes of lipids. However, simultaneous analysis of lipid and fatty acid compositions to both temperature and growth phase has been rarely investigated. Temperature has a major effect on the composition of fatty acids produced by microalgae. Most microalgal species respond to increased growth temperature by decreasing the ratio of unsaturated to saturated fatty acids [158]. As per Table 19, in all cases, monounsaturated fatty acids increased while polyunsaturated fatty acids were down-regulated when the culture temperature increased beyond the optimal temperature. Meanwhile, in most cases, the content of saturated fatty acids increased with temperature and vice versa. Suga et al [159] discussed that low temperatures induced the expression of genes encoding fatty acid desaturates, including those that were able to desaturate oleic acid (18:1) to linoleic acid (18:2) or linoleic acid (18:2) to linolenic acid (18:3). In other words, lower growth temperature causes microalgae to generate higher content of lipid with a high percentage of unsaturated fatty acids, which helps the strain to maintain membrane fluidity under low temperatures [151]. This is however inconsistent with the observations of some researchers in the cases of Nannochloropsis oculata and Chlorella vulgaris, which found that the percentage of saturated fatty acid decreased when the culture temperature increased beyond the optimal temperature [46]. However, The response of microalga chemical composition to low and high growth temperatures is speciesdependent with no overall consistent relationship between temperature

Table 18 The effect of culture temperature on microalgae lipid, lipid productivity and growth rate. Temp °C

Worst Temp

Best Temp

Negative or No effect

Algal Species

L-LC

H-LC

L-LP

H-LP

L-B g L-1

H-B g L-1

day

Ref

10–45

35–45

20

Ch. minutissima

17.5%

14%

–

–

1.5B

9B

–

[35]

25–38 18 &26 15–25

38 26 15

25 18 25

> < > – <

1x 1x 1x

1.3x 1.2x 1.07x

− 2.72 0.030 gld 9.11

20.22 0.023 gld 10.10

– – –

– – –

14 1026C− 1518C 14

[46] [151] [46]

10–30 25–35 25–35 25–35 25–35 25–35 15&30 15–35

10 33 30 33 30 33 15 15

20 27 25 27 25 25 30 30

> > > > > > – <

Ch. vulgaris Ch. sorokiniana Nannochloropsis. oculata Scenedesmus sp. Isochrysis sp. Chaetoceros sp. Rhodomonas sp. Cryptomonas sp prymnesiophyte Isochrysis galbana Nannochloropsis salina

1xTAG 20.2% 12.2 8.0 19.6 13.8 19% 45%

1.83xTAG 21.7% 16.8 12.7 21.4 14.7 24% 57%

– – – – – –

– – – – – –

0.31 gld

0.43 gld

1x – – – – – 0.1B 0.4B

1.56x – – – – – 0.5B 0.5B

15 – – – – – 6 10

[152] [158] [158] [158] [158] [158] [150] [28]

30 15 25 25 20 27 25 27 25 25 25–30 <

L: Lowest, H: Highest, LC: Lipid Content, LP: Lipid productivity, GR: Growth Rate, B: Biomass Production, Worst and best conditions in terms of Lipid content and Lipid Productivity.

225

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– – – – – – – – – – – – – – –

~ ~ – – – – – –

– – –

↑↑ ↑ ~ ↑ – ↓ ↑↓ ↑ ↑ ↑ ↓ ↓ – – – ~ – – – –

↑ ↑ ~ ↑ ~ ↑

~ ↓

– – –

– – – ~ – – – –

– – – –

– – b c – b – –

– – – – – ↑↓ ↑↓ ↓ – ↑

– – ~ – ↓ – ↓ ~ ↑ ↑ – ↑ ↑ ↑ ↓ ↑ ↓ ↓

– – ↑ – ↓ ↑ ~ ~ ↑↓ – – – ↑↑ ↑

– – ↓ – – ~↑ ↑ ~ ↑↓ – – – ↓↓ ↓

– – – – –

– – – – – – ↓ – ~ – – – – –

– – – – –

↑↑ ↑↑ ~ ↑↑ ↑↓ ~ ↑ ↑ ~ ↑ ↓

↓ ↓ ↑ ↑ ↑↓ ↑ ↑ ~ ↑↓ ↑ ↑ ↑ ↑↑ ↑ ↓

↓ ↓ ↓ ↓↓ ↑↓ ~ ↑↓ – ↑ ↓ – ~ ↓↓ ↓ ↓

– – – – –

– – – a –

↓↓ ↓ ↑ – ↓ ↑ ↑↑ ↑ ↑ ↑ – ↓ ~ ↑ ↑ ↓ ↑ ↓

↑↑ ↑↑ ↑ ↑↑ ↑ ↑↓ ↑ ↑ ↑ ↑ – ↑ ↑ ↑ ↓ ↑ ~ ~

Earth receives approximately 3.9 × 106 MJ of solar energy each year, however, only a small portion of this radiation is converted to biomass and chemical energy. Algae are photosynthetic organisms and hence light is a vital source for their photosynthetic activity and autotrophic growth. However, microalgae are only capable of using narrow wavelengths (400–700 nm ~ 43% of the total solar energy) of the natural light spectrum. Hence, utilization of suitable light wavelengths and light intensity are the other key factors that can affect or even control the biomass and lipid production in algae. Generally, photosynthesis incorporates light-dependent reactions and dark reactions. In the former phase, light energy is captured and converted to energy carriers and oxygen is released as a by-product of photolysis. The electrons provided by the oxygen during light reactions are transferred to two large proteins including PSI and photosystems II (PSII) [160–162]. Dark reactions, also referred to as the Calvin-Benson (CB) cycle, occur concurrently with the light-dependent reactions and do not require light. The three phase of CB cycle include CO2 fixation, reduction, and regeneration which are associated with thirteen different enzymatic reactions. Excess light intensities causes photo-oxidation to PSII components and the subsequent photo-inhibition. This phenomenon damages the essential electron transferor proteins during photosynthesis and finally reduces microalgal productivity [163,164]. Generally, high light intensities results in photoinhibition at the initial growth stages, while low light intensities are insufficient for cell growth at the next stages of cultivation (due to self-shading effect). Hence, gradual increase of light intensity till its optimum value improves growth rate. The light-dark (L-D) cycle is the other important factor, which affects the growth rate of microalgae. The biochemical composition of algae is altered by change of light/dark cycle. As a result, enhanced frequencies of the light-dark cycles can dramatically increase the photosynthetic efficiency and microalgal biomass productivity and vice versa [165]. Wahidin et al. [166] found that the growth rate and lipid content of Nannochloropsis sp. in batch cultivation increased by changing the L-D cycle from 12:12–18:06 h. The authors reported that longer light irradiation was better at high light intensities (200 µmol m2 -1 s ) while shorter light irradiation was better at moderate light intensities (100 µmol m-2 s-1). Generally, microalgal biomass production can increase under red light irradiation while the optimal light wavelength for lipid and carotenoid accumulations are blue and far-red light wavelengths. Chlorophyll a and b are the major light harvesting pigments. These pigments and the algae containing these pigments are sensitive to wavelengths of blue and red light. As an example, green algae, which contain chlorophyll a and b, grow better in blue and red light. In order to enhance microalgal productivity, light wavelength, intensity and light-dark cycles should be adjusted to achieve an optimal balance between photoprotection and photosynthesis. This strategy would also allow for a maximum utilization of natural light irradiation by microalgae. There are two comprehensive review articles on the effect of light on the growth of different algae species [167] and the strategies to improve microalgae productivity by using light irradiation [162].

– – – –

– – – ↓ ~ ~ ~ ~ ~

–

A: C22:3, ↑↓,b: C20:5: ↓, c C22:6: ↓↓, bC20:5: ↓↓, *eukaryoticalgae, **.

↓↓ ↓ ↑ ~ ↑ ↑ ↑ ↑ ↑ ↑ – ↑ ~ ↑ ↑ ↓ ↑ ↓ – – – – – – – – –

– – – – – – ↑ ~ – ~ – – ~ ↑

– – ↑ – ↑↓ ↑ ↑ ~ ↑ ↑ – ↑↓ ~ ↑ ~ ↓ ~ ~ Chlorella. vulgaris Nannochloropsis. oculata Chlorella sorokiniana Scenedesmus sp. Isochrysis sp. Chaetoceros sp. Rhodomonas sp. Cryptomonas sp prymnesiophyte Isochrysis galbana Selenastrum capricornutum Phaeodactylum tricornutum Chlorella vulgaris* Botryococcus braunii* Spirulina platensis* * Spirulina platensis* Chlorella vulgaris* Botryococcus braunii*

25 25 26 20 20 27 30 25 25–27

↑25–38 ↑15–25 ↑18–26 ↑10–30 ↑25–30 ↑25–35 ↑25–33 ↑25–30 ↑25–30 ↑15–30 10–25 10–25 20–30 18–32 30&40 30&40 20&30 18&32

– – – – – – – – – – – – ~ ~

C 15:1 C 15:0 C 14:0

C 14:1

and fatty acid unsaturation [158]. 5.8. Light effects

T Edible Vegetable Oil

Table 19 The effect of temperature on fatty acid composition of different microalgae.

C 16:0

C 16:1

C 16:2

C 16:3

C 16:4

C 17:0

C 17:1

C 18: 0

C 18:1

C 18:2

C 18:3

C 20:0

C 20:1

C 20:2

SFA

MUFA

PUFA

Ref

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6. Simulation and modeling of microalgae behavior The increasing rate of microalgae-based technologies and applications has encouraged the development of new mathematical models to study the simultaneous effect of different factors and variables that influence microalgae growth and allow forecasting of algal production. Studies on modeling of microalgae growth kinetics were initiated with the work by Droop [168,169]. After that, a number of models have been developed based on single factors such as nitrogen [170], temperature 226

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[171], light intensity [172], and photosynthesis and photoinhibition effects [173]. Contrary to other factors (e.g. pH, temperature and nutrient levels) which can be adjusted and maintained to avoid limiting or inhibitory effects, the light intensity cannot be easily controlled at its optimum level. Hence, many researchers have focused on modeling the behavior of biomass as a function of light intensity. Moreover, these models are mainly steady-state, which keep the factors values constant over time. In most recent works, the studies have been focused on more complicated dynamic models that can predict the interactive effect of multi-parameters (e.g. light intensity and nitrogen [174], temperature, light intensity and pH [175]), following a structure according to Monod’s or Droop's kinetics models. In contrast to steady-state models, factor values change over time in dynamic models. On the other side, the interactions between microalgae and bacteria have also been extensively studied and modeled. Research on the growth of microalgae and bacteria in HRAPs started with the pioneering work by Buhr and Miller [176]. Since then more complicated models have been developed such as River Water Quality Model 1 (RWQM1) [177] or Sah Model [178]. RWQM1 models the growth of microalgae and bacteria on nitrogen (N) and phosphorous (P), but it does not include inorganic carbon (C) limiting growth. In Sah model, the effects of nutrients, environmental factors (i.e. temperature, solar radiation, and wind) and physical processes (such as re-aeration) are taken into account. These mechanistic models are mainly based on Monod kinetics. It should also be noted that most of the integrated microalgae-bacteria models do not combine the overall biochemical processes and the simultaneous effects of pH, temperature, light intensity, or high dissolved oxygen (DO) concentration on biomass growth. This weakness has been well-addressed in one of the new integral mechanistic model BIO-ALGAE [179].

achieved and supplied, will be the ultimate goal of biofuel production from microalgae. This work presented a comprehensive review on the mutual and individual effects of nutrients (e.g. nitrogen, phosphorus, iron) and environmental stress (e.g. pH, temperature, NaCl, CO2) on microalgae lipid content, productivity, growth rate and lipid composition. It summarizes the lipid compositions of the most suitable and widely studied strains as well as the effect of environmental factors on their lipid compositions. Although most researchers have focused on individual effects of the aforementioned parameters, getting benefits from synergistic or antagonistic effects of those factors can lead us toward the best economic strategies to reach the highest lipid productivity. It has been found that nitrogen deprivation with iron supporting can be one of the most effective strategies. Temperature, though is very vital for microalgal life, does not have significant effects on lipid increment. Increment in temperature can even be lethal for microalgae. The other useful strategy is to support the microalgae to reach their maximum density (growth) and apply the environmental stress at the stationary phase. On the other hand, addition of organic carbon sources greatly increases the growth rate of microalgae and the lipid content of some microalgae. Optimized value of carbon source combined with low-nutrition condition greatly improves the microalgae production efficiency and shortens the cultivation cycle.

7. Biotechnological engineering of microalgae

References

Acknowledgement The authors are grateful to the financial support of the National Science Foundation (NSF EPSCoR RII Grant No. OIA-1632899). Various other supports from the University of Mississippi are also gratefully acknowledged.

Biotechnological engineering of microalgae aims at enhancing the lipid content of microalgae which can be achieved by either conventional, genetic engineering and metabolic engineering approaches. Conventional methods include nutrient deprivation, physical stress like temperature, salt stress, and heavy metal stresses etc., as already discussed. In recent years, there has been an increasing interest and expectation on the genetic engineering of microalgae for the generation of strains with high productivity of either fermentable carbohydrates or fatty acids. Improving photosynthetic efficiency by increasing light penetration and decreasing cell shading, engineering and improving different enzymes toward lipid biogenesis and identifying rate-limiting enzymes/committed step are among the most important strategies that have been studied to reach the full potential of microalgae. However, most of such studies have been limited to a few species. Instability and low efficiency of transgenes expression, as well as the lack of suitable promoters, are the main difficulties preventing nuclear transformation of new microalgal strains. Moreover, the use of microalgae as platforms to produce recombinant proteins and the efficient engineering of metabolic pathways has been hampered in some cases due to low expression of exogenous genesis. Hence, it is necessary to develop new tools, which ensure high expression levels and stability of the transgene [1,180].

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